Methods of Detecting Alloantibodies Using HLA and Non-HLA Antigens

ABSTRACT

Described herein are materials and methods for detecting alloantibodies using both HLA and non-HLA antigens in a single assay.

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/222,614, filed on Sep. 23, 2015, which is incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to materials and methods for detectingalloantibodies using both HLA and non-HLA antigens.

BACKGROUND

Transplant rejection occurs when the immune system of the recipient of atransplant, particularly antibodies produced by the recipient, attacksthe transplanted organ or tissue. The recipient's immune systemrecognizes the transplanted organ as foreign tissue and attempts todestroy it. Rejection also occurs when the transplanted organ comprisesthe donor's lymphocytes or progenitor stem cells, which may generate animmune response to the recipient tissues such as graft vs. host disease.Chronic rejection is a term used to describe all long term loss offunction in organ transplants associated with chronic alloreactiveimmune response. Long term chronic rejection usually leads to a need fora new transplanted organ about a decade after the initial transplant.Human leukocyte antigens (HLA) are one type of molecules within atransplanted organ in which the recipient's immune system attacks thatcauses a transplant rejection.

It is a standard practice in the transplant field to test all potentialrecipients against a panel of HLA antigens selected to represent a humanpopulation and the percentage of HLA alleles against which the serum isreactive is determined. In this panel reactive antibody (PRA) testingreaction of a patient's serum against a high percentage of HLA allelespresent in a normal human population is predictive of a high risk ofgraft rejection.

Alloantibodies, particularly when donor specific, are one of the mostimportant factors that cause both early and late graft rejection.Despite improvements in the transplantation outcomes, antibody-mediatedrejection (AMR) remains substantial and it is associated with increasedmorbidity, mortality and costs (Colvin, ASN 18(4):1046-1056, 2007).

The presence of HLA antibodies is widely believed to be the majorelements contributing to humoral graft rejections. Transplant recipientswith high panel reaction antigens (PRA) are associated with early graftrejection. Elevated donor specific HLA antibodies in the organrecipients either before and/or after allograft transplantation has beenassociated with acute and chronic AMR and decreased long term graftsurvival. Despite advanced HLA typing matching programs, there has notbeen a major improvement in the incidence of Graft-versus-host disease(GVHD). Several studies indicate that in addition to HLA alloantibodies,transplant recipients also develope antibodies against antigens otherthan HLA molecules. The role of alloantibodies against non-HLA antigensis a critical element in the pathogenesis of acute and chronic allograftoutcomes (Tinckam and Chandraker, CJASN 1(3):404-414, 2006).

Currently, there are no defined non-HLA alloantibody antigens. MHC classI-related chain A (MicA), a group of polymorphic non-HLA antigensexpressed on endothelial cells, have been implicated in the pathogenesisof hyperacute, acute and chronic organ allograft rejections(Sumitran-Holgers son, Current Opinion Immunology. 20(5):607-13, 2008).In addition, Vimentin, Angiotensin II Type I receptor (AT1R), LG3peptide of Perlecan and Collagen V are also considered to be non-HLAantigens (Sigdel and Sarwal, Human Immunology, 74:1486-1490, 2013).Targets for anti-endothelial cell antibody (AECA), islet cell antibodies(ICAs), anti-Liver sinusoidal endothelial cells (anti-LSECs) andAntineutrophil cytoplasmic autoantibodies (ANCA) are also considered asnon-HLA antigens. The AECA, ICA, anti-LSECs and ANCA target antigens arenot well defined (Hepatology, 40(5):1211-1221, 2004).

Accordingly, there remains a need in the art for improved methods of HLAtyping including methods for determination of percentage of PRA which israpid, convenient and accurate.

SUMMARY

In one aspect, described herein is a composition comprising a firstcollection of solid-phase substrates each coated with different purifiedhuman leukocyte antigens (HLAs) to represent the HLA antigen populationof a single cell line and a second collection of solid-phase substratescoated with a different non-HLA antigen listed in Table 1 or Table 1A.In some embodiments, the different purified HLA antigens are Class I HLAantigens. In some embodiments, the different purified HLA antigens areClass II HLA antigens.

In some embodiments, the first collection comprises 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, 54 55, 56, 57, 58, 59, 60 or moredifferent Class I HLA antigens. In some embodiments, the firstcollection comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more differentClass II HLA antigens. In some embodiments, the first collectioncomprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 5455, 56, 57, 58, 59, 60 or more different Class I HLA antigens and orClass II HLA antigens.

In some embodiments, the second collection comprises the non-HLA antigenset forth in Table 1 or 1A. In some embodiments, the second collectioncomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-HLA antigensset forth in Table 1 and/or Table 1A.

In another aspect, described herein is a kit for determining thepercentage of panel reactive antibodies in serum of a subject againstHLA antigens comprising a first collection of solid-phase substrateswherein each solid-phase substrate is coated with different purified HLAantigens to represent the HLA antigen population of a single cell linesuch that said collection simulates the distribution of HLA antigens ina normal human population and a second collection of solid phasesubstrates wherein each substrate is coated with different purifiednon-HLA antigens listed in Table 1 or Table 1A.

In another aspect, described herein is a method for determining thepercentage of panel reactive antibodies in serum of a subject againsthuman leukocyte antigens (HLA) antigens, said method comprising:contacting a first collection of solid-phase substrates subtypes and asecond collection of solid-phase substrate subtypes with serum from saidsubject for a sufficient time for anti-HLA antibodies in said serum tobind to said HLA-antigens to form a complex, wherein each substratesubtype in the first collection is coated with different purified HLAantigens to present HLA antigens derived from a cell population of asingle cell, wherein each substrate subtype of the second collection iscoated with different purified non-HLA antigens listed in Table 1 orTable 1A, detecting the presence of the complex to determine thepresence or absence of panel reactive antibodies, and determining thepercentage of panel reactive antibodies in the serum. In someembodiments, the subject is a transplant or transfusion recipient. Insome embodiments, the serum sample is collected before the subject hasreceived a transplant or transfusion. In other embodiments, the serumsample is collected after the subject has received a transplant ortransfusion. In further embodiments, the serum sample is collected bothbefore and after the transplant or transfusion.

The method of determining the percentage of panel reactive antibodiesmay be carried out to monitor the risk that the recipient will rejectthe transplant or transfusion or develop graft versus host disease(GVHD). Thus, in one embodiment the method may further comprise the stepof obtaining a base line percentage of panel reactive antibodies beforethe subject receives the transplant or transfusion. The methods may alsocomprise a step of comparing the percent of panel reactive antibodiesbefore and after receipt of the transplant and transfusion. Themonitoring may be carried out at various time points, after transplantor transfusion to determine if the subject is developing GVHD. Forexample, in some embodiments, the baseline percentage of panel reactiveantibodies is determined between a time period ranging from 1 hour toabout 1 year or longer before the subject receives the transplant ortransfusion. In some embodiments, the baseline percentage of panelreactive antibodies is determined about 1 hour, about 6 hours, about 12hours, about 1 day, about 5 days, about 1 week, about 2 weeks, about 3weeks, about 4 weeks, about 1 months, about 2 months, about 3 months,about 4 months, about 5 months, about 6 months, about 7 months, about 8months, about 9 months, about 10 months, about 11 months about 1 year orlonger before the subject received the transplant or transfusion. Insome embodiments, the percent of panel reactive antibodies is determinedbetween a time period ranging from 1 hours to about 1 year or longerafter the subject has received the transplant or transfusion. Forexample, in some embodiments, the percent of panel reactive antibodiesis determined about 1 hour, about 6 hours, about 12 hours, about 1 day,about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks,about 1 months, about 2 months, about 3 months, about 4 months, about 5months, about 6 months, about 7 months, about 8 months, about 9 months,about 10 months, about 11 months about 1 year or longer after thesubject has received the transplant or transfusion.

In some embodiments, the detecting step comprises detecting labeledligand bound to the complex to determine the presence or absence ofpanel reactive antibodies. In some embodiments, detecting of the labeledligand is carried out by flow cytometry. In some embodiments, thedetecting step comprises detecting the presence of the complex using asolid phase immunoassay or a multiplexed bead immunoassay.

The solid-phase substrate can be any solid substrate known in the art.In some embodiments, the solid-phase substrate is selected from thegroup consisting of microparticle, microbead, magnetic bead, ion torrentbead, flow cytometer bead and an affinity purification column. In someembodiments, the solid-phase substrate is a microbead. In someembodiments, the microbead is a laytex microbead. The microbead, in someembodiments, has a diameter ranging from about 2 μm to about 15 μm,inclusive. Microbeads having a diameter of about 2 μm, 3 μm, 4 μm, 5 μm,6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm or 15 μm arealso contemplated. In some embodiments, at least one microbeadpresenting Class I HLA antigens is 3 μm is diameter. In someembodiments, at least one microbead presented Class II HLA antigens is 5μm in diameter. In some embodiments, the microbeads comprise a mixtureof 3 μm microbeads presenting Class I HLA antigens and 5 μm microbeadspresented Class II HLA antigens.

In some embodiments, each solid phase substrate is detectablydistinguishable from other solid phase substrates within thecomposition. In some embodiments, the detectably distinguishable solidphase substrates are distinguishable by fluorescent labels.

In some embodiments, the different purified HLA antigens are Class I HLAantigens. In some embodiments, the HLA antigens are selected such thatthe HLA antigens presented on the solid phase substrate comprise Class IHLA antigens so as to simulate the distribution of Class I HLA antigensin a normal human population. In some embodiments, the first collectioncomprises 54 different Class I HLA antigens. In some embodiments, the 54different Class I HLA antigens are purified from 30 different celllines. In some embodiments, the first collection comprises 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 55, 56, 57, 58, 59, 60 ormore different Class I HLA antigens.

In some embodiments, the different purified HLA antigens are Class IIHLA antigens. In some embodiments, the first collection comprises 22different Class II HLA antigens. In some embodiments, the firstcollection comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more differentClass II HLA antigens.

In some embodiments, the second collection comprises different non-HLAantigens set forth in Table 1 or 1A. In some embodiments, the secondcollection comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more non-HLAantigens set forth in Table 1 and/or Table 1A. The non-HLA antigen, insome embodiments, is a fusion protein comprising at least one domain,wherein the domain is a signal peptide, a modified cytoplasmic domain,purification tag or detection tag. In some embodiments, the domain isthe B2 signal peptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPDTag.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 describes the output data from the non-HLA multiplex assay. Foursubjects (S10823K, S11114A, S11143B and FL71681) show distinct crossrelativities against a panel of non-HLA antigens.

FIG. 2 describes the trend line of increase of anti-non-HLA antibodiesin samples obtained from a patient undergoing 2nd lung graft in amultiplex platform.

FIG. 3 describes (a) the fusion DPD tag that enhances the multiplexreactivity and (b) the structure of DPD indicates it is an alpha helixloop.

FIG. 4 describes the Box and whiskers' plot of non-HLA antigens from twopatient population using a panel of 21 non-HLA antigens in one test.

FIG. 5 describe the reactivity of non-HLA fusion proteins detected by ananti- fusion antibody on a multiplex platform.

FIG. 6 depicts the correlation between the results of the method of theinvention in determining the percentage PRA versus a standard cytotoxitytest for sample sera.

FIGS. 7A-7D depict the reaction of the mixture of Class I and Class IIbeads and their reaction to anti-HLA Class I antibodies (FIGS. 7A and7B) or anti-HLA Class II antibodies (FIGS. 7C and 7D).

DETAILED DESCRIPTION

Graft rejection is when transplanted tissue is rejected by therecipient's immune system, which destroys the transplanted tissue. Therejection is an adaptive immune response via cellular immunity andhumoral immunity. Chronic rejection induced by humoral response is amajor cause of graft dysfunction and re-transplantation. It is wellrecognized that pre-existing antibodies to HLA antigens expressed by theallograft is detrimental to survival of allografts. The presence ofpanel-reactive antibodies (PRA) against HLA antigens beforetransplantation can lead to early rejection. Despite intensive HLAtyping screening for HLA matching and progressive monitoring thedevelopment of anti-HLA alloantibodies, declining graft function remainsa paramount clinical concern.

With the focus on the graft rejection among HLA-identical-siblingrecipients, the slow decline in survival curves of HLA-identical-siblingtransplants suggests that antigens other than HLA antigens maycontribute to allograft rejection. Transplant recipients have alsodeveloped antibodies against targets other than HLA molecules (non-HLAantigens), such as autoimmune antigens.

The development of a solid phase platform as described in the Examplesprovided herein allows for large-scale antibody screening for both HLAand non-HLA antigens. As described in Example 6, antibodies tokidney-expressed non-HLA antigens have been identified in kidneyallografts patients, resulting in acute kidney rejection and allograftloss. Autoantibodies are also found in patients with chronic humoralrejection.

The present inventors have discovered that the currently availablemethods useful for detecting HLA antibodies are not reliable andreproducible for the detection of non-HLA antibodies. For example, ELISAis the most common detection method. However, it is more suitable for asingle target. Flow cytometry against panels of endothelial cells isanother method, but the use of a cell based assay may result in highbackground and reliability reduced. As non-HLA antibodies become morerelevant to antibody-mediated processes, development of reproducibleassays on the multiplex global scales optimized for transplantation forthese antibodies becomes important. The multi-plex assays describedherein provide a reliable and reproducible method for determining thepresence of both HLA and non-HLA antibodies in a single assay.

HLA Antigens

The HLA locus is highly polymorphic in nature. As disclosed in theNomenclature for Factors of the HLA System 2000 (Hum. Immunol.;62(4):419-68, 2001) there are 124 HLA-A alleles, 258 HLA-B alleles, 74HLA-C alleles, 221 HLA-DRB 1 alleles, 19 DRB3 alleles, 89 DRB4 alleles,14 DRB5 alleles, 19 DQA1 alleles and 39 DQB1 alleles, with new allelesbeing discovered continuously. As testament to this rapid progress, aApril 2007 update by the WHO nomenclature Committee for Factors of theHLA System (www.anthonynolan.com/HIG/) showed there are 545 HLA-Aalleles, 895 HLA-B alleles, 307 HLA-C alleles, 8 HLA-E alleles, 12 HLA-Halleles, 9 HLA-J alleles, 6 HLA-K alleles, 4 HLA-L alleles, 4 HLA-Palleles, 3 HLA-V alleles, 3 DRA alleles, 494 DRB 1 alleles, 1 DRB2alleles, 44 DRB3 alleles, 13 DRB4 alleles, 18 DRB5 alleles, 3 DRB6alleles, 2 DRB7 alleles, 10 DRB8 alleles, 1 DRB9 alleles, 34 DQA1alleles, 83 DQB1 alleles, 23 DPA1, 126 DPB1 alleles, 4 DMA alleles, 7DMB alleles, 12 DOA alleles and 9 DOB alleles.

Solid phase immunoassays for the detection and characterization ofHLA-specific antibodies provide increased sensitivity and specificity,while being more efficient for time and, compared to the traditionallyused cell-based methods. Multiplexed bead immunoassay (MBIA) has emergedas a powerful tool to simultaneously detect several antibodies targetsin limited sample volumes. The limited sample volume and time-savinggains of the MBIA have made it an election technique for studiesinvolving multiple factors. The invention provided herein allows for themultiplexed bead immunoassays detecting both HLA reactive antibodies andnon-HLA reactive antibodies in a single assay.

Non-HLA Antigens

This invention describes the development of a multiplex solid phaseplatform allowing for global-scale antibody screening for both HLA andnon-HLA antigens in a biological sample. For example, using suchmethods, in some embodiments, antibodies to kidney-expressed non-HLAantigens in the kidney allografts patients can be monitored. Sometransplant recipients develop autoantibodies with acute kidney rejectionand allograft loss. Autoantibodies are also found in patients withchronic humoral rejection.

The targets of humoral responses against non-HLA antigens are primarilyantigens expressed on endothelial cells and epithelial cells andcategorized as non-HLA alloantigens or tissue-specific autoantigens.Most of them are either patient- or graft-specific. Whether antibodiesto non-HLA antigens are pathogenic and/or whether they can be used asbiomarkers for transplant outcome remains unclear (J Am Soc Nephrol 22:1168-1178, 2011). In addition, targets for anti-endothelial cellantibody (AECA), islet cell antibodies (ICAs), anti-Liver sinusoidalendothelial cells (anti-LSECs) and antineutrophil cytoplasmicautoantibodies (ANCA) are considered as non-HLA antigens. The AECA, ICA,anti-LSECs and ANCA target antigens are not defined and are subject toevery research lab's definition (Hepatology, 40(5):1211-1221, 2004).

Agrin is the most abundantly expressed glycoprotein in the glomerularbasement membrane (GBM). The GBM is a basement membrane specialized inultrafiltration and consists of various matrix molecules, includingfibronectin, and collagens. The 22 kDa C-terminus Agrin fagment (CAF) isrecently discovered as the biomarkers for kidney function and physicalhealth activities (American journal of nephrology, 38(6):501-508). Thepresence of anti-Argrin antibodies was associated with the number ofrejection episodes prior to diagnosis of transplant glomerulopathy(TGP), a symptom of kidney failure after kidney transplant (AmericanJournal of Transplantation 2005; 5: 383-393).

Angiotensinogen (AGT) is a component of the renin-angiotensin system(RAS), a hormone system that regulates blood pressure and fluid balance.It is also known as the renin substrate, and is a non-inhibitory memberof the serpin family of proteinase inhibitors. It causesvasoconstriction and a subsequent increase in blood pressure. AGT hasshown very strong correlation in renal graft rejection and has beenvalidated by customized ELISA assays in independent patient sera andtheir localization confirmed by immunohistochemistry (J. Proteome Res.,2010, 9 (12), pp 6715-6721).

Angiotensin II type 1 receptor (AT1R, or ATGR1) is a G protein-coupledreceptor that mediates angiotensin effects and causes vasoconstrictionin vascular smooth muscle. It mediates most physiologic andpathophysiologic actions of its endogenous ligand, angiotensin II, withoveractivity leading to vascular remodeling and hypertension. Antibodiesto AT1R are implicated in several vascular pathologies. Several studieshave shown that AT1R is associated with antibody-mediated organrejection.

Rho GDP-dissociation inhibitor 2 is a protein that, in humans, isencoded by the ARHGDIB gene. It regulates the GDP/GTP exchange reactionof the Rho proteins by inhibiting the dissociation of GDP from them, andthe subsequent binding of GTP to them. By using two-dimensional Westernblotting experiments, Rho GDP-dissociation inhibitor has been identifiedas the non-HLA antigens target in patients undergoing chronichemodialysis.

Aurora kinase A-interacting protein (AURKA) is a cell cycle-regulatedkinase that appears to be involved in microtubule formation and/orstabilization at the spindle pole during chromosome segregation. AURKAprotein is found at the centrosome in interphase cells and at thespindle poles in mitosis. This gene may play a role in tumor developmentand progression. By comparing antibody repertoires in pre- andpost-transplant sera from several cohorts of patients with and withouttransplant glomerulopathy, de novo increase of anti-AURKA has beenidentified as non-HLA antigen.

Complement C4-B is a part of the classical activation pathway. Itprovides a surface for interaction between the antigen-antibody complexand other complement components. It can be cleaved to release C4anaphylatoxin, a mediator of local inflammation. Deficiency of thisprotein is associated with systemic lupus erythematosus. C4B has beeninvolved with graft injuries by combined with C2a and starts cascadesreactions in the antibody mediated damages.

Chromatin assembly factor 1 subunit B (CHAF1b, CAF-1, or p60) isrequired for the assembly of histone octamers onto newly-replicated DNA.CAF-I is composed of three protein subunits, p50, p60, and p150. Theprotein encoded by this gene corresponds to the p60 subunit and isrequired for chromatin assembly after replication. The encoded proteinis differentially phosphorylated in a cell cycle-dependent manner. Inaddition, it is normally found in the nucleus except during mitosis,when it is released into the cytoplasm. CHAF1b-specific antibodies werepredominantly detected in patients with acute myeloid leukemia (AML) oneyear after allogeneic bone marrow transplantation.

CXCL11 is a small cytokine belonging to the CC chemokine family. Geneexpression of CXCL11 is strongly induced by IFN-γ and IFN-β, and weaklyinduced by IFN-αCXCL11 has been identified independently as I-TAC.CXCL11 is thought to play a critical role in allograft rejection. It isa dominant chemokine in controlling skin intragraft inflammation. Byusing high-density protein arrays to identify non-HLA antibodies inchronic allograft injury (CAI) and subsequently validated a subset in acohort of 172 serum samples collected serially post-transplantation, theauthors have identified CXCL11 as the non-HLA antigens (Sigdel et al.Non-HLA antibodies to immunogenic epitopes predict the evolution ofchronic renal allograft injury. JASN Apr. 1, 2012 vol. 23 no. 4750-763).

CXCL9, also known as MIG, is a CXC inflammatory chemokine. CXCL9 plays akey role in leukocyte trafficking and induces angiostatic effects inhuman microvacular endothelial cells. CXCL9 enhances T lymphocytefunction in alloimmune response. CXCL9 is induced by cytokines,particularly IFNγ during infection, injury, or immunoinflammatoryresponses. Similar to CXCL11, CLCX9 was identified as non-HLA in thebone marrow transplant patients by microarray.

Cyclophilin A or peptidylprolyl isomerase A (PPIA) is a ubiquitouslydistributed protein belonging to the immunophilin family. PPIA wasinitially believed to function primarily as an intracellular protein.Recent studies have revealed that it can be secreted by cells inresponse to inflammatory stimuli. It has shown that extracellular PPIAstimulates pro-inflammatory signals in endothelial cells (EC) andvascular smooth muscle cells (VSMC). Similar to AURKA, by compareantibody repertoires in pre- and post-transplant sera from severalcohorts of patients with and without transplant glomerulopathy, de novoincrease of anti-PPIA has been identified as non-HLA antigen (Dinavah etal., Antibodies Reactive to Non-HLA Antigens in TransplantGlomerulopathy, J Am Soc Nephrol 22: 1168-1178, 2011).

Eukaryotic translation initiation factor 2A (eIF2A) is a eukaryoticinitiation factor. It is required in the initiation of translation. Itis an essential factor for protein synthesis. Since eIF2 is essentialfor translation initiation and therefore protein synthesis, defects ineIF2 are lethal. Its activity is regulated by a mechanism involving bothguanine nucleotide exchange and phosphorylation. By using IgG isolatedfrom patients with allograft rejection and look the reactivity againstendothelial cell surface, eIF2A has been identified an antigen ofinterests for liver transplant allograft rejection.

Alpha-enolase (EOS-1), also known as phosphopyruvate hydratase, isresponsible for the catalysis of the conversion of 2-phosphoglycerate(2-PG) to phosphoenolpyruvate (PEP), the ninth and penultimate step ofglycolysis. Higher concentrations of ENO-1 in cerebrospinal fluid morestrongly correlated to low-grade astrocytoma. Increased levels of alphaenolase have also been identified in patients who have suffered a recentmyocardial infarction or cerebrovascular accident. By looking for theAnti Endothelial Cells antibody targets in the anti-neutrophilcytoplasmic antigens (ANA) associated vasculitides, ENO-1 has beenidentified as one of the targets.

Glutamate decarboxylase 2 or glutamic acid decarboxylase 2 (GAD2, GAD65)is an enzyme that catalyzes the decarboxylation of glutamate. It is thetargets of autoantibodies in people who later develop type 1 diabetesmellitus or latent autoimmune diabetes. Autoimmunity is the term todescribe an attack against native cells and tissues by the immunesystem. An autoimmune response against glutamic acid decarboxylase inneurons has been implicated in a rare neurological condition known asStiff-Man syndrome. It has been proposed that a similar autoimmuneresponse against GAD in pancreatic cells may be associated with type 1diabetes. Circulating GAD65 can be used as a biomarker of islet damageor transplant rejection and it will facilitate in vivo studies of thepathogenesis of anti-GAD65 autoreactivity. By checking simultaneouspancreas-kidney transplant (SPKT) recipients on type 1 diabeticpatients, the incidence of rejection episodes was significantly higherin pretransplantation GAD autoantibody-positive daclizumab-treatedrecipients compared with GAD autoantibody-negative or ATG-treatedrecipients (Janet al., Pretransplantation GAD-Autoantibody Status toGuide Prophylactic Antibody Induction Therapy in Simultaneous Pancreasand Kidney Transplantation. Transplantation 96(8):745-752, 2013).

Glial cell-derived neurotrophic factor, also known as GDNF is a smallprotein that potently promotes the survival of many types of neurons.GDNF has regenerative properties for brain cells and showed potential astreatment for Parkinson's disease—monkeys with an induced form ofParkinson's disease showed less trembling when treated with the drug,and neuronal fibres grew in part of the human brain exposed to the drug.Similar to CXCL11, GDNF is identified as non-HLA in the bone marrowtransplant patients by microarray in the chronical renal graftrejection.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is involved inseveral steps of gene expression regulation. It integrates cellularsignaling cascades with multiple processes of gene expressionmechanisms. This protein has a role during cell cycle progession of geneexpression. It is one of the major pre-mRNA-binding proteins. HNRNPK hasbeen reported to be involved in the life cycle of different viruses byeither direct interaction with viral proteins. hnRPNK has beenidentified by screen a coronary artery cells cDNA library againstcardiac allograft vasculopathy patient serum sample as the new antigenictargets (Acevedo et al., Antibodies against heterogeneous nuclearribonucleoprotein K in patients with cardiac allograft vasculopathy.Journal of Heart and Lung Transplantation, 30(9):1051-1059, 2011).

Intercellular adhesion molecule 1, ICAM-1, also known as CD54, binds toCD11a/CD18 (HNA5), or CD11b/CD18 (HNA4), and is known for its importancein stabilizing cell-cell interactions and facilitating leukocyteendothelial transmigration. More recently, ICAM-1 has been characterizedas a site for the cellular entry of human rhinovirus. Signal-transducingfunctions of ICAM-1 seem to be associated primarily with proinflammatorypathways. In particular, ICAM-1 signaling seems to produce a recruitmentof inflammatory immune cells such as macrophages and granulocytes.ICAM-1 is considered one of AECA. 60% of cardiac recipients havedeveloped anti ICAM-1 IgM (Lawson et al., Anti-intercellular adhesionmolecule-1 antibodies in sera of heart transplant recipients: a role inendothelial cell activation. Transplantation 2005; 80: 264-271).

Gamma-interferon inducible protein 16 (IFI16) also known asinterferon-inducible myeloid differentiation transcriptional activator.IFI16 has been shown to play a role in the sensing of intracellularDNA—and has also been linked to HIV-infected helper T-cell pyroptosis.IF116 binds nuclear viral DNA, triggering expression of antiviralcytokines in response to infection with herpesviruses. Similar to eIF2A,IgG isolated from patients with allograft rejection reacts againstendothelial cell surface. IF116 has been identified as an antigen ofinterest for liver transplant allograft rejection.

Interferon-gamma (IFN-gamma) is crucial for immunity againstintracellular pathogens and for tumor control. However, aberrantIFN-gamma expression has been associated with a number ofautoinflammatory and autoimmune diseases. It is a potent activator ofmacrophages, which has antiproliferative effects on transformed cellsand can potentiate the antiviral and antitumor effects of the type Iinterferons. IFN-gamma is produced mainly by T-cells and natural killercells activated by antigens, mitogens, or alloantigens. Similar toCXCL11, IFN-gamma is identified as non-HLA in the bone marrow transplantpatients by high density microarray in the chronical renal graftrejection.

The interleukin-2 receptor (IL-2R) is a heterotrimeric protein expressedon the surface of certain immune cells, such as lymphocytes, that bindsand responds to a cytokine called IL-2. It has three subunits, generatedby different combinations of three different proteins, often referred toas “chains”: α (alpha) (also called IL-2Rα, CD25, or Tac antigen), β(beta) (also called IL-2Rβ, or CD122), and γ (gamma) (also calledIL-2Rγ, γc, common gamma chain, or CD132); these subunits are also partsof receptors for other cytokines. The β and γ chains of the IL-2R ismembranes of the type I cytokine receptor family. IL-2 and its receptorhave key roles in key functions of the immune system, tolerance andimmunity, primarily via their direct effects on T cells. Thepolymorphism of alpha chain has been reported linked to multiplesclerosis, an autoimmune disease.

Interleukin-7 receptor subunit alpha (IL7R-a), also known as CD127, isthe alpha-subunit of IL7 Receptor for interleukin-7 and acts as areceptor for thymic stromal lymphopoietin (TSLP). The interleukin-7receptor a chain transmits distinct signals for proliferation anddifferentiation during B lymphopoiesis and is essential for thedevelopment of T Cells. There are reports indicating IL7R polymorphismsis associated with inflammatory demyelinating diseases.

Insulin (INS) is a peptide hormone produced by beta cells in thepancreas and it regulates the metabolism of carbohydrates and fats bypromoting the absorption of glucose from the blood to skeletal musclesand fat tissue. Insulin also inhibits the production of glucose by theliver. Type 1 diabetes is a chronic illness characterized by the body'sinability to produce insulin due to the autoimmune destruction of thebeta cells in the pancreas. Anti-insulin antibodies are a cause ofhypoglycemia following pancreas transplantation. In Islet celltransplantation for the treatment of Type 1 diabetes, insulinautoantibodies can be detected in Type 1 diabetes.

Far upstream element-binding protein 2 (FUBP2) binds to the dendritictargeting element and may play a role in mRNA trafficking. It mayactivate gene expression. FUBP2 represents a novel and frequentpro-tumorigenic mechanism promoting proliferation (tumor growth) andmotility (dissemination) of human liver cancer cells. Similar to ENO-1,by looking for the Anti Endothelial Cells antibody targets in theanti-neutrophil cytoplasmic antigens (ANA) associated vasculitides,FUBP2 has been identified as one of the AECA targets.

Lamins are components of the nuclear lamina, a fibrous layer on thenucleoplasmic side of the inner nuclear membrane, which is thought toprovide a framework for the nuclear envelope and may also interact withchromatin. Lamin A and C are present in equal amounts in the lamina ofmammals. Lamin-A plays an important role in nuclear assembly, chromatinorganization, nuclear membrane and telomere dynamics. Whereas Lamin-B 1(LMNB 1) (˜585 aa) is in the protein matrix over inner nuclear membraneand has been associated with aging. LMNB 1 forms homodimer. There is acommon polymorphism A510V (2%) in Lamin-B1, which is not thatsignificant. The only commercial source for LMNB1 protein is from wheatgerm in vitro translation system. There are some LMNB 1 Elisa kitsavailable but it aims for the antigen detection rather than forautoimmune rejection. Lamin A has been identified as one of targetantigens of anti-endothelial cell and anti-vascular smooth muscle cellantibodies in patients with giant cell arteritis. Similar to RhoGDP-dissociation inhibitor, by using two-dimensional Western blottingexperiments, Lamin B has been identified as the non-HLA antigens targetin patients undergoing chronic hemodialysis.

Myosin comprise a family of ATP-dependent motor proteins and are bestknown for their role in muscle contraction and their involvement in awide range of other eukaryotic motility processes. Cardiac myosin (CM)is a heart specific antigen implicated in allograft rejection.Pretransplant myosin autoantibodies correlated with acute cardiactransplant rejection. The expansion of alloreactive T cells was followedby an increase of cardiac myosin reactive T cells and development ofanti-myosin IgG1 autoantibodies in a mouse heart transplant modelmismatched for minor histocompatibility alloantigens. This supports theidea that CM released during alloimmune injury of the allograft isrecognized by CD4+ T helper autoreactive cells through indirectrecognition pathway and triggers the generation of autoreactive CMantibodies. Notably, mature CM is not expressed in the thymus duringdevelopment which may result in incomplete negative selection (Zhang andReed, Non-MHC antigenic targets of the humoral immune response intransplantation. Curr Opin Immunol. 2010 October; 22(5): 682-688.).Since CM consists of myosin heavy chain and light chains, they may alsoresponsible for the graft rejections.

Neuropilin-1 (NRP-1) bind many ligands and various types ofco-receptors; they affect cell survival, migration, and attraction. Someof the ligands and co-receptors bound by neuropilins are vascularendothelial growth factor (VEGF) and semaphorin family members. It is amembrane-bound coreceptor to a tyrosine kinase receptor. Neuropilinexpression is up-regulated in multiple tumor types, and correlates withtumor progression and prognosis in specific tumors. Neuropilins mayindirectly mediate effects on tumor progression by affectingangiogenesis or directly through effects on tumor cells. (Bates et al.,High diversity of non-human leukocyte antigens in transplant-associatedcoronary artery disease. Transplantation. 2003; 75:1347-1350.)

Nuclear and spindle-associated protein 1 (NuSap1) has been reported tofunction in mitotic spindle assembly, chromosome segregation, andregulation of cytokinesis. Depletion of NUSAP1 from cells led to thesuppression of double strand DNA break repair via the homologousrecombination and single-strand annealing pathways. NUSAP1 has recentlybeen identified as a biomarker for aggressive prostate cancer. Bytesting the sera on protein array, Nusap1 is identified as one of thetargets of de novo antibody after allogeneic allogeneic hematopoieticcell transplantation (HCT), Wadia et al., Antibodies specifically targetAML antigen NuSAP1 after allogeneic bone marrow transplantation. Blood.115(10): 2077-2087 2010.)

Collagen V (Col V) acts as a major risk factor after human lungtransplantation. Col V is not normally expressed in healthy tissue.However, Col V is unveiled during graft injuries in lung transplants.Col V-specific T cells appear in lung transplant recipients before theclinical onset of rejection. It has been implicated in a number ofautoimmune or inflammatory conditions and allograft rejection. CollagenV are associated with chronic rejection after lung transplantation(American Journal of Transplantation 2014; 14: 685-693).

The ErbB3 binding protein-1 (EBP1) or Proliferation-associated protein2G4 (PA2G4) belongs to a family of DNA/RNA binding proteins implicatedin cell growth, apoptosis and differentiation. Ebp1 is a well-conservedDNA/RNA binding protein that is implicated in cell growth, apoptosis anddifferentiation in many cell types. Similar to eIF2A, IgG isolated frompatients with allograft rejection reacts against endothelial cellsurface. EBP1 has been Identified an antigen of interests for livertransplant allograft rejection.

Peroxiredoxin 2 (PRDX2) might participate in the signaling cascades ofgrowth factors and tumor necrosis factor-alpha by regulating theintracellular concentrations of H2O2. It is the third most abundantprotein in erythrocytes. PRDX2 is an essential antioxidant enzyme thatprevents the oxidative inactivation of VEGF receptor-2 in vascularendothelial cells. Proteins extracted from human umbilical veinendothelial cells (HUVEC) were separated by two-dimensionalelectrophoresis, and Western blotting was subsequently conducted usingsera from patients with systemic vasculitis.PRDX2 has been identified asone of the anti-endothelial cell antibodies (AECA) targets in systemicvasculitis (Karasawa et al., Peroxiredoxin 2 is a novel autoantigen foranti-endothelial cell antibodies in systemic vasculitis. Clin ExpImmunol. 161(3):459-70), 2010.

Protein Kinase C-zeta plays an important role in insulin-stimulatedglucose transport. It has at least two alternative transcripts, thefull-length PKCζ (this protein) and an N-terminal truncated form PKMζ.PKCζ is about 67 kDa (592 aa) and located in the cytoplasmic region.

BPI fold-containing family A member 1 (BPIFA1), or palate, lung andnasal epithelium clone (PLUNC) plays a role in the innate immuneresponses of the upper airways. It reduces the surface tension insecretions from airway epithelia and inhibits the formation of biofilmby pathogenic Gram-negative bacteria. BPIFA1 binds bacteriallipopolysaccharide (LPS) and negatively regulates airway surface liquidhomeostasis and proper clearance of mucus. It plays a role in the airwayinflammatory response after exposure to irritants.

26S protease regulatory subunit 6B (PSMC4) is involved in theATP-dependent degradation of ubiquitinated proteins. The regulatory (orATPase) complex confers ATP dependency and substrate specificity to the26S complex. It is a part of the immunoproteasome whose function is toprocess class I HLA peptides. PSMC4 has been shown to interact with anorphan member of the nuclear hormone receptor superfamily highlyexpressed in liver, and with gankyrin, a liver oncoprotein. Similar toPLUNC, PSMC4 is discovered as the one of the non-HLA antibodies targetsfrom the lung transplants.

Islet cell antigen 512 also termed IA-2 is a novel autoantigen of type 1diabetes, which has a tyrosine phosphatase-like domain. IA-2 is a majortarget of islet cell autoantibodies. The frequencies of autoantibodiesagainst glutamic acid decarboxylase 65 (GAD65) and islet cell antigen(ICA) 512/IA-2 (512/IA-2) are common on the specific human leukocyteantigen (HLA) in type 1 diabetes mellitus (T1D). In pancreastransplantations, anti-GAD (Glutamic Acid Decarboxylase) and anti-IA2(protein tyrosine phosphatase, IA-2) autoantibodies is related to theonset of rejection or graft loss. In islet transplantation, the presenceof autoantibodies also correlates with a worse evolution and could be akey factor in the chronic failure of the graft (Diabetol Metab Syndr.2009; 1: 9).

Tyrosine-protein phosphatase non-receptor type 22 (PTPN22) affects theresponsiveness of T and B cell receptors, and mutations are associatedwith increases or decreases in risks of autoimmune diseases. PTPN22 genehas been associated with autoimmune disorders, including an increasedrisk of Type 1 Diabetes, rheumatoid arthritis, Systemic LupusErythematosus (SLE), Vitiligo and Graves' disease, but a decreased riskof Crohn's disease. PTPN22 acts as negative regulator of T-cell receptor(TCR) signaling by direct dephosphorylation of the Src family kinasesLCK and FYN, ITAMs of the TCRz/CD3 complex, as well as ZAP70, VAV, VCPand other key signaling molecules. Protein tyrosine phosphatasenon-receptor 22 (PTPN22) plays a central role in T cell, B cell andinnate immune cell signaling. The allelic polymorphism, TPN22R620W-variant allele, could be involved in the susceptibility to acuteallograft rejection in kidney transplant patients (Transplant Proc. 2009March; 41(2):657-9). By using PTPN22 knockout mouse, the lack of theprotein tyrosine phosphatase PTPN22 improves transplant tolerance topancreatic islets in mice (Diabetologia. 2015 Mar. 7).

Ribosomal Protein L7 (RPL7) plays a regulatory role in the translationapparatus. It is located in the cytoplasm. RPL7 has been shown to be anautoantigen in patients with systemic autoimmune diseases, such assystemic lupus erythematosus. By screening of a HUVEC cDNA library withtransplant-associated coronary artery disease sera, RPL7is identified asa candidate autoantigen associated with transplant rejection. (Clin ExpImmunol. 2001; 126:173-179).

Speedy Homologue A (SPDYA) as a member of the Speedy/RINGO family and anovel activator of cyclin-dependent kinases, was shown to promote cellcycle progression and cell survival in response to DNA damage. SPDYA isa cell cycle protein that promotes cell proliferation by activatingcyclin-dependent kinase-2 (CDK2; 116953) at the G1/S phase transition.Overexpression of SPDYA in several human and mouse cell lines increasedDNA replication and the rate of cell proliferation. Similar to AGT,SPYDA has shown very strong correlation in renal graft rejection and hasbeen validated by customized ELISA assays in independent patient seraand their localization confirmed by immunohistochemistry.

Tumor necrosis factor alpha (TNF, tumor necrosis factor, TNFα, cachexin,or cachectin) is a cell signaling protein (cytokine) involved insystemic inflammation. The primary role of TNF is in the regulation ofimmune cells. TNF, being an endogenous pyrogen, is able to induce fever,apoptotic cell death, cachexia, inflammation and to inhibittumorigenesis and viral replication. TNFα stimulates IL1 and GM-CSF,increases tissue damage by IL1 and induces the onset of collagenases byfibroblasts and chondrocytes. It has a role in modulating HLA class 2expression, as well as the adhesion molecule. TNF α level more than 45pg/mL can be taken as an immunological marker of renal transplantrejection (Saudi J Kidney Dis Transpl 2009; 20(6):1000-1004).

Regenerating Islet-derived protein 3-alpha (Reg3A) orpancreatitis-associated protein 1 (PAP1) is a pancreatic secretoryprotein that may be involved in cell proliferation or differentiation.PAP is activated in primary liver cancers. Elevation of PAP in patientswith pancreatic cancer is not merely explainable by concomitantpancreatitis, but seems to be due to increased PAP production by thecancer cells. Elevated anti Reg3A s has been reported on simultaneouskidney-pancreas transplantation (SKP Tx) patients (2015 AmericanTransplant Congress Abstract #446).

Receptor tyrosine-protein kinase ERBB-3, also known as HERS (humanepidermal growth factor receptor 3), is a member of the epidermal growthfactor receptor (EGFR/ERBB) family of receptor tyrosine kinases. Thekinase-impaired ERBB3 is known to form active heterodimers with othermembers of the ErbB family, most notably the ligand binding-impairedERBB2. ERBB3 binds to the ligands heregulin and NRG-2 and causes achange in conformation that allows for dimerization, phosphorylation,and activation of signal transduction. Similar to eIF2A, IgG isolatedfrom patients with allograft rejection reacts against endothelial cellsurface. ERBB3 has been Identified an antigen of interests for livertransplant allograft rejection.

Platelet glycoprotein 4, or CD36, also known as FAT (fatty acidtranslocase), FAT/CD36, (FAT)/CD36, SCARB3, GP88, glycoprotein IV(gpIV), and glycoprotein Mb (gpIIIb), is an integral membrane proteinfound on the surface. CD36 interacts with a number of ligands, includingcollagen types I and IV, thrombospondin, erythrocytes,platelet-agglutinating protein p37, and long-chain fatty acids. CD36function in long-chain fatty acid uptake. CD36 is recognized as Nakaantigen. The abnormality of anti CD36 antibody has been linked to heartfailure in transplant coronary artery disease (Int J Mol Med. 1998 June;1(6):1007-10).

Nucleolin (NCL) is a multifunctional phosphoprotein ubiquitouslydistributed in the nucleolus, nucleus and cytoplasm of the cell. NCL isa eukaryotic nucleolar phosphoprotein, involved in the synthesis andmaturation of ribosomes. NCL may play a role in the process oftranscriptional elongation. It regulates various aspects of DNA and RNAmetabolism, chromatin structure, rDNA transcription, rRNA maturation,cytokinesis, nucleogenesis, cell proliferation and growth, the folding,maturation and ribosome assembly and nucleocytoplasmic transport ofnewly synthesized pre-RNA. Antibodies against NCL are found in manytransplant patients and they seemed to be associated with kidneyallografts rejection and with coronary artery disease in hearttransplant recipients (Transplantation 2011;92: 829-835).

Peroxisomal trans-2-enoyl-CoA reductase, PECR, is an enzyme responsiblefor the reduction of phytenoyl-CoA to phytanoyl-CoA in peroxisomes. PECRis strongly expressed in the kidney. Recently, it has been shown thatanti-PECR antibodies could be associated with transplant glomerulopathy.Similar to AURKA, by compare antibody repertoires in pre- andpost-transplant sera from several cohorts of patients with and withouttransplant glomerulopathy, de novo increase of anti-PECR has beenidentified as a non-HLA antigen.

E3 ubiquitin-protein ligase TRIM21, also known as Tripartitemotif-containing protein 21 (TRIM21) is an intracellular antibodyeffector in the intracellular antibody-mediated proteolysis pathway.TRIM21, also known as Ro52 is often the target of circulatingautoantibodies in autoimmune diseases. Studies showed that anti-Ro52antibodies are associated with different clinical outcomesTRIM21 is partof the RoSSA ribonucleoprotein, which includes a single polypeptide andone of four small RNA molecules. It interacts with autoantigens inpatients with Sjogren's syndrome and systemic lupus erythematosus.TRIM21 is considered as one of AECA target protein. High anti-TRIM21 wascorrelated to renal rejection.

Proteasome subunit alpha type-4, PSMA4, is a multicatalytic proteinasecomplex which is characterized by its ability to cleave peptides withArg, Phe, Tyr, Leu, and Glu adjacent to the leaving group at neutral orslightly basic pH. Proteasome dysfunction leads to many diseasesincluding cancer, and drugs that inhibit proteasome activity directlyaffect lung cancer susceptibility through its modulation of cellproliferation and apoptosis. It has been reported that proteasomesubunit alpha type-4 (PSMA4) mRNA levels are increased in lung tumors,and down-regulation of PSMA4 expression decreased proteasome activity.PSMA4 has been identified on renal rejection patient as one of thenon-HLA candidates (American Journal of Transplantation 2009;9:2126-2135).

Tissue factor (F3) also called platelet tissue factor, factor III,thromboplastin, or CD142 is a protein present in subendothelial tissueand leukocytes necessary for the initiation of thrombin formation fromthe zymogen prothrombin. The best known function of tissue factor is itsrole in blood coagulation. The signaling function of F3 plays a role inangiogenesis and apoptosis. Similar to PLUNC, F3 is discovered as theone of the non-HLA antibodies targets from the lung transplants.

60 kDa SS-A/Ro ribonucleoprotein is also known as TROVE domain family,member 2 (TROVE2) functions as a RNA chaperone that binds to misfoldedpre-5S ribosomal RNA and may hasten the degradation of the defectivemolecule. Autoantibodies directed against Ro/SSA and La/SSB autoantigenswere originally identified in patients with Sjögren's syndrome andsystemic lupus erythematosus (SLE). Subsequent studies showed thatanti-Ro/SSA antibodies may be present in patients with other autoimmunediseases, including systemic sclerosis, idiopathic inflammatorymyopathies (IIM), primary biliary cirrhosis (PBC), and rheumatoidarthritis (RA). Additionally, anti-Ro/SSA antibodies (with or withoutanti-La/SSB antibodies) identify pregnant women who are at increasedrisk of having a child with neonatal lupus syndrome. Polymorphism ofTROVE2 (L10P) has linked to lung transplant by TGF-beta (Clin RevAllergy Immunol. 2011 February; 40(1): 27-41).

Interferon-induced helicase C domain-containing protein 1 (IFIH1) playsa major role in sensing viral infection and in the activation of acascade of antiviral responses including the induction of type Iinterferons and proinflammatory cytokines. IFIH1 polymorphisms have beenassociated with type 1 diabetes. Autoimmune disease risk variant ofIFIH1 is associated with increased sensitivity to IFN-α and serologicautoimmunity in lupus patients (J Immunol. 2011 Aug. 1;187(3):1298-303).

Tubulin is the major building block of microtubules. The tubulin familyconsist of alpha- and beta-tubulin. To form microtubules, the dimers ofα- and β-tubulin bind to GTP and assemble onto the (+) ends ofmicrotubules while in the GTP-bound state. Antibodies to KA1 tubulin(TUBA1B) is associated with chronic rejection after lung transplantation(J Immunol. 2008 Apr. 1; 180(7):4487-94). Similar to RhoGDP-dissociation inhibitor, by using two-dimensional Western blottingexperiments, beta tubulin has been identified as the non-HLA antigenstarget in patients undergoing chronic hemodialysis.

Perlecan (PLC) also known as basement membrane-specific heparan sulfateproteoglycan core protein (HSPG) or heparan sulfate proteoglycan 2(HSPG2). Perlican LG3 peptide lies inside the Endorepellin subunit whichis the domain V of Perlecan. LG3 is a biomarker for breast cancer, IgAinduced nephropathy, physical status, and acute allograft vascularrejection. Patients with increased anti-LG3 antibodies have correlatedwith accelerated organ rejection. In addition, anti-LG3 antibodies alsoincrease deposit buildups and induce clogged arteries (American Journalof Transplantation 2013; 13: 861-874).

PRKR-interacting protein 1 protein (PRKRIP1) binds double-stranded RNA.PRKRIP1 interacts with PKR (protein kinase RNA-activated) and functionsto inhibit or negatively regulate PKR activity and is associated withadipogenesis. Similar to AGT, PRKRIP1 has shown very strong correlationin renal graft rejection and has been validated by customized ELISAassays in independent patient sera and their localization confirmed byimmunohistochemistry.

Endothelin receptor type A, also known as ETAR or EDNRA, is a human Gprotein-coupled receptor for the endothelin-1. Endothelin-1 promotesmyofibroblast induction through the ETA receptor via arac/phosphoinositide 3-kinase/Akt-dependent pathway and it is essentialfor the enhanced contractile phenotype of fibrotic fibroblasts. ENDRApolymorphism 1136L has been linked to breast cancer. EDNRA expressesonly in platelets. The presence of anti-ETAR antibodies is associatedwith a decrease renal transplant function during the first 12monthsafter transplantation (Transpl Immunol. 2014 Jan.; 30(1):24-9).

Fibronectin (FN) is a high-molecular weight (˜440 kDa) glycoprotein ofthe extracellular matrix that binds to membrane-spanning receptorproteins called integrins. FN plays a major role in cell adhesion,growth, migration, and differentiation, and it is important forprocesses such as wound healing and embryonic development. Similar toCollagen V, elevated anti FN antibodies has linked to transplantGlomerulopathy in renal graft recipients (American Journal ofTransplantation 2014; 14: 685-693).

Fibronectin Leucine-rich Repeat transmembrane protein 2 (FLRT2)functions in cell adhesion and receptor signaling. FLRT2 is required inthe epicardium to promote heart morphogenesis. FLRT2 is involved inmediating cell-matrix interactions. Anti-FLRT2 antibody has thepotential to induce direct endothelial cell cytotoxicity. By using thehuman umbilical vein endothelial cells retroviral expression system,FLRT2 has been identified as one of the AECA targets on for systemiclupus erythematosus patient (Arthritis Res Ther. 2012; 14(4): R157).

Vimentin (VIM) is a non-polymorphic intermediate filament expressed incytosol of endothelial, vascular smooth muscle cells, activatedplatelets and macrophages, renal tubular cells, mesangial cells andrenal stromal cells. VIM expressed in the intima and media of coronaryarteries where vascular smooth muscle cells and fibroblasts locate.Autoimmune responses to VIM are associated with both acute and chronicrejection of heart and renal allografts. Anti-vimentin antibodies are anindependent predictor of transplant-associated coronary artery diseaseand can be used to identify some of the patients who are at high risk ofdeveloping this complication (Transplatation Vol. 71, 886-892, No. 7,Apr. 15, 200).

Glutathione S-transferase theta-1 (GSTT1) conjugate reduced glutathioneto a wide number of exogenous and endogenous hydrophobic electrophiles.Individuals with a homozygous deletion of the glutathione S-transferasetheta 1 (GSTT1) gene lack GSTT1 enzymatic detoxification and have highrisk of acute myeloid leukemia. In liver transplant,antibodies againstglutathione-S-transferaseT1 (GSTT1) expressed on the graft may induce anantibody response leading to a severe graft dysfunction. In addition,donor- specific antibodies against MICA and GSTT1 antigens could beresponsible for the occurrence of antibody-mediated kidney graftrejection (Transplantation 2009; 87: 94-99).

Endoplasmic reticulum lipid raft-associated protein 2 (ERLIN2) plays acritical role in inositol 1,4,5-trisphosphate (IP3) signaling bymediating ER-associated degradation of activated IP3 receptors.Mutations in this gene are a cause of spastic paraplegia-18 (SPG18).ERLIN2 is in the prohibitin family of proteins that definelipid-raft-like domains of the ER. ERLIN2 may confer a selective growthadvantage for breast cancer cells by facilitating a cytoprotectiveresponse to various cellular stresses. Similar to eIF2A, IgG isolatedfrom patients with allograft rejection reacts against endothelial cellsurface. ERLIN2 has been Identified an antigen of interests for livertransplant allograft rejection.

Complement Factor H (CFH) is a member of the regulators of complementactivation family. Factor H has been shown to interact with Complementcomponent 3. A shortage (deficiency) of complement factor H can causeuncontrolled activation of the complement system. Complement factor Hdeficiency, a known hereditary risk factor for post-transplantthrombotic microangiopathy (TMA), may also favor development of acuteallograft glomerulopathy AAG. Unopposed complement activation is a riskfactor for both immune and nonimmune forms of microvascular injuries inrenal allografts (Fortin et al. Am J Transplant. 2004 Feb.; 4(2):270-3.)Atypical Hemolytic Uremic Syndrom (HUS) associated with anti-CFHautoantibodies is an uncommon illness associated with high risk ofprogression to end-stage renal disease (Khandelwal et al. PediatrTransplant. 2014 August; 18(5):E134-9)

Complement C3 produced within the kidney is an important mediator ofinflammatory and immunological injury. Synthesis of complement componentC3 regulates acute renal transplant rejection. Patients with SLE hadincreased titers of anti-C3 antibodies, compared with healthy controls.C3 nephritic factors (increased C3 autoantibodies) prolong the half-lifeor prevent regulation of the alternative pathway C3 convertase; resultin uncontrolled complement activation. They are strongly associated withrenal disease with symptoms like acquired partial lipodystrophy (APLD)or C3 glomerulopathy (C3GP) (Dragon-Durey 2013, Molecular Immunology 56(2013) 213-221)

Phospholipase A2 Receptor , a 185 kDa type I transmembrane glycoproteinexpressed on glomerular podocytes, is identified as a major targetantigen of the autoantibodies involved in membranous nephropathy (MN), acommon cause of adult nephrotic syndrome, one of the most commonglomerulonephritides involving the renal transplant. (Dai et al. 2015,Nature). Idiopathic membranous nephropathy, a common form of thenephrotic syndrome, is an antibody-mediated autoimmune glomerulardisease. A majority of patients with idiopathic membranous nephropathyhave antibodies against a conformation-dependent epitope in PLA2R. PLA2Ris present in normal podocytes and in immune deposits in patients withidiopathic membranous nephropathy, indicating that PLA2R is a majorantigen in this disease. In addition, Anti-PLA(2)R autoantibodies inserum samples from patients with membranous nephropathy were mainlyIgG4.

In some embodiments, the non-HLA antigen is selected from the set ofnon-HLA antigens set forth in Table 1.

TABLE 1 Uniprot SEQ Non-HLA Antigen Description Alias Access No. ID NOAgrin (CAF) AGRN O00468 1 Angiotensinogen AGT P01019 2 RhoGDP-dissociation inhibitor 2 ARGHDIB P52566 3 Aurora kinaseA-interacting protein AURKA Q9NWT8 4 Complement C4-B C4B, C4D P0C0L5 5Chromatin assembly factor 1 subunit B CHAF1b, CAF-1, p60 Q13112 6 C—X—Cmotif chemokine 11 ITAC, CXCL11 O14625 7 C—X—C motif chemokine 9 MIG,CXCL9 Q07325 8 Cyclophilin A PPIA P62937 9 Eukaryotic translationinitiation factor 2A EIF2A Q9BY44 10 Alpha-enolase ENO1 P06733 11Glutamate decarboxylase 2 GAD2, GAD65 Q05329 12 Glial cell line-derivedneurotrophic factor GDNF P39905 13 Heterogeneous nuclearribonucleoprotein K HNRNPK P61978 14 Intercellular adhesion molecule 1ICAM-1, CD54 P05362 15 gamma-interferon inducible protein 16 IFI16Q16666 16 gamma-interferon IFN-γ P17803 17 Interleukin-2 receptorsubunit alpha IL2RA, CD25 P01589 18 Interleukin-7 receptor subunit alphaIL7R, CD127 P16871 19 Insulin INS P01308 20 Far upstream element-bindingprotein 2 FUBP2, KHSRP Q92945 21 Lamin A/C LMNA P02545 22 Lamin B1 LMNB1P20700 23 NEUROPHILIN-1 NRP1, CD304 O14786 24 Nucleolar andspindle-associated protein 1 NUSAP1 Q9BXS6 25 ERBB3 Binding protein 1PA2G4, EBP1 Q9UQ80 26 PEROXIREDOXIN 2 PRDX2 P32119 27 Protein KinaseC-zeta PKC-Z Q05513 28 BPI fold-containing family A member 1 PLUNC,BPIFA1 Q9NP55 29 26S protease regulatory subunit 6B PSMC4 P43686 30Islet cell antigen 512 PTPRN, PTPIA2, ICA512 Q16849 31 Tyrosine-proteinphosphatase non-receptor type 22 PTPN22 Q9Y2R2 32 Ribosomal Protein L7RPL7 P18124 33 Speedy Homologue A SPDYA Q5MJ70 34 Tumour necrosis factoralpha TNF-α P01375 35 Regenerating Islet-derived protein 3-alpha PAP-1,REG3A Q06141 36 Receptor tyrosine-protein kinase erbB-3 ERBB3 P21860 37Platelet glycoprotein 4 CD36 P16671 38 Nucleolin NCL P19338 39Peroxisomal trans-2-enoyl-CoA reductase PECR Q9BY49 40 E3ubiquitin-protein ligase TRIM21 TRIM21, RO52 P19474 41 Proteasomesubunit alpha type-4 PSMA4 P25789 42 Tissue factor F3, TFA, CD142 P1372643 60 kDa SS-A/Ro ribonucleoprotein TROVE2, RO60 P10155 44Interferon-induced helicase C domain-containing IFIH1 Q9BYX4 45 protein1 alpha Tubulin-1A TUBA1A Q71U36 46 alpha Tubulin 1B TUBA1B P68363 47alpha Tubulin 1C TUBA1C Q9BQE3 48 beta Tubulin TUBB P07437 49 PerlecanLG3 HSPG2 P98160 50 PRKR-interacting protein 1 PRKRIP1 Q9H875 51Endothelin Receptor type A EDNRA, ETAR P25101 52 FibronectinLeucine-rich Repeat Transmembrane FLRT2 O43155 53 protein Vimentin VimP08670 54 Angiotensin II Type I receptor AT1R, AGTR1 P30556 55 C-typelectin domain family 16, member A CLEC16A Q2KHT3 56 Collagen I COL1A1,COL1A2 P02452 57 Collagen II COL2A1, COL2A2 P02458 58 Collagen IIICOL3A1, COL3A2 P02461 59 Collagen IV COL4A1, COL4A2 P02462 60 Collagen VCOL5A1, COL5A2 P20908 61 Cytotoxic T-lymphocyte protein 4 CTLA4, CD152P16410 62 Endoplasmic reticulum lipid raft-associated protein 2 ERLIN2O94905 63 Fibronectin FN1 P02751 64 Glutathione S-transferase theta-1GSTT1 P30711 65 Keratin, type II cytoskeletal 1 KRT1 P04264 66 MyosinHeavy Chain alpha MYH6 MYH6 P13533 67 Myosin Heavy Chain beta MYH7 MYH7P12883 68 Myosin Light Chain MYL4 MYL4 P12829 69 Zinc finger protein 33AZNF33A Q06730 70 Zinc transporter 8 ZnT8, SLC30A8 Q8IWU4 71 ComplementFactor H CHF P08603 72 Complement C3 C3 P01024 73 Phospholipase A2Receptor PLA2R1 Q13018 74

In some embodiments, the non-HLA antigen is selected from the set ofnon-HLA antigens set forth in Table 1A.

TABLE 1A Uniprot Non-HLA Antigen Description Alias Access No.Publication No. Protein Kinase C-zeta PKC-Z Q05513 US 20120077689Ribosomal Protein L7 RPL7 P18124 U.S. Pat. No. 7,132,245 Perlecan LG3HSPG2 P98160 US20130004978 Endothelin Receptor type A EDNRA, ETAR P25101U.S. Pat. No. 8,592,164 Vimentin Vim P08670 U.S. Pat. No. 7,132,245Angiotensin II Type I receptor AT1R, AGTR1 P30556 U.S. Pat. No.8,425,877 Collagen II COL2A1, COL2A2 P02458 WO2000037940 Collagen VCOL5A1, COL5A2 P20908 U.S. Pat. No. 8,039,225 Glutathione S-transferasetheta-1 GSTT1 P30711 US 20110039281 Myosin Light Chain MYL4 MYL4 P12829US 20120077689 Zinc transporter 8 ZnT8, SLC30A8 Q8IWU4 US20100143374Complement Factor H CFH P08603 U.S. Pat. No. 8,501,427Anti-Phospholipase-A2-Receptor PLA2R1 Q13018 US 20110177534

Preparation of HLA and Non-HLA-Antigens

In some embodiments, the HLA antigen and/or the non-HLA antigen is afusion protein. For example, the invention provides for transforming ortransfecting host cells with a nucleic acid encoding the amino acidsequence of an HLA antigen polypeptide or a non-HLA antigen polypeptidefused with a heterologous domain selected from the group consisting ofB2 signal peptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag. Anucleic acid molecule encoding the amino acid sequence of an HLA antigenpolypeptide or a non-HLA antigen polypeptide may be fused with thedomain and inserted into an appropriate expression vector using standardligation techniques. Exemplary vectors include, but are not limited to,bacterial vectors, eukaryotic vectors, plasmids, cosmids, viral vectors,adenovirus vectors and adenovirus associated vectors.

The HLA antigen polypeptide and/or the non-HLA antigen polypeptide maycontain a sequence encoding a “tag” or exogenous amino acid sequence,such as an oligonucleotide molecule located at the 5′ or 3′ end of thenon-HLA polypeptide coding sequence; an oligonucleotide sequenceencoding polyHis (such as hexaHis), FLAG, hemaglutinin influenza virus(HA), V5 or myc or other tags, for which commercially availableantibodies exist. This tag may be fused to the non-HLA polypeptide uponexpression. The term “exogenous” as used herein refers to a substance ormolecule originating or produced outside of an organism. The term“exogenous gene” or “exogenous nucleic acid molecule,” as used herein,refers to a nucleic acid that codes for the expression of an RNA and/orprotein that has been introduced (“transformed”) into a cell or aprogenitor of the cell. An exogenous gene may be from a differentspecies (and so a “heterologous” gene) or from the same species (and soa “homologous” gene), relative to the cell being transformed.

In some embodiments, the expression vectors contain sequences forcloning and expression of exogenous nucleotide sequences. Such sequencesmay include one or more of the following nucleotide sequences: apromoter, one or more enhancer sequences, an origin of replication, atranscriptional termination sequence, a complete intron sequencecontaining a donor and acceptor splice site, a sequence encoding aleader sequence for polypeptide secretion, a ribosome binding site, apolyadenylation sequence, a polylinker region for inserting the nucleicacid encoding the polypeptide to be expressed, and a selectable markerelement.

In some embodiments, the vector comprises a selectable marker geneelement. A selectable marker gene element encoding a protein necessaryfor the survival and growth of a host cell grown in a selective culturemedium may also be a component of the expression vector. Exemplaryselection marker genes include those that encode proteins thatcomplement auxotrophic deficiencies of the cell; or supply criticalnutrients not available from complex media. The invention alsocontemplates that the HLA antigen polypeptides and/or non-HLA antigenpolypeptides described herein comprise one or more of these exogenousamino acid sequences.

In some embodiments, a leader, or signal, sequence is used to direct thenon-HLA antigen polypeptide (or HLA antigen polypeptide) out of the stemcell after administration. For example, a nucleotide sequence encodingthe signal sequence is positioned in the coding region of the non-HLAantigen encoding nucleic acid (or HLA antigen encoding nucleic acid), ordirectly at the 5′ end of the non-HLA antigen coding region (or HLAantigen coding region). The signal sequence may be homologous orheterologous to the non-HLA antigen polypeptide (or HLA antigenpolypeptide) gene or cDNA, or chemically synthesized. The secretion ofthe non-HLA antigen polypeptide (or HLA antigen polypeptide) from thestem cell via the presence of a signal peptide may result in the removalof the signal peptide from the secreted non-HLA antigen polypeptide (orHLA antigen polypeptide). The signal sequence may be a component of thevector, or it may be a part of the nucleic acid molecule encoding thenon-HLA antigen polypeptide (or HLA antigen polypeptide) that isinserted into the vector.

In some embodiments, the domain is a cytoplasmic domain, or trafficsignal, sequence. Cytoplasmic domain sequences may be used to direct thenon-HLA antigen polypeptides (or HLA antigen polypeptides) out of thecells after administration or to modify its characteristics to avoidcell signaling pathway that leads to cell death.

The vectors described herein optionally comprise a promoter operablylinked to the nucleic acid encoding the non-HLA antigen polypeptide (orHLA antigen polypeptide). Promoters are untranscribed sequences locatedupstream to the start codon of a structural gene that control thetranscription of the structural gene. Inducible promoters initiateincreased levels of transcription from DNA under their control inresponse to some change in culture conditions, such as the presence orabsence of a nutrient or a change in temperature. Alternatively,constitutive promoters initiate continual gene product production withlittle or no control over gene expression. A large number of promoters,recognized by a variety of potential host cells, are well known. Thenative non-HLA (or HLA) gene promoter sequence may be used to directamplification and/or expression of the non-HLA (or HLA) polypeptidenucleic acid molecule. A heterologous promoter also may be used toinduce greater transcription and higher yields of the non-HLA (or HLA)polypeptide expression as compared to the non-HLA (or HLA) polypeptideexpression induced by the native promoter.

In addition, an enhancer sequence may be inserted into the vector toincrease the transcription of a DNA encoding the non-HLA antigenpolypeptide (or HLA antigen polypeptide). Enhancers are cis-actingelements of DNA, usually about 10-300 by in length, that act on thepromoter to increase transcription. Enhancer sequences available frommammalian genes include globin, elastase, albumin, alpha-feto-proteinand insulin. Exemplary viral enhancers that activate eukaryoticpromoters include the SV40 enhancer, the cytomegalovirus early promoterenhancer, the polyoma enhancer, and adenovirus enhancers. While anenhancer may be spliced into the vector at a position 5′ or 3′ to anucleic acid molecule encoding the non-HLA antigen polypeptide (or HLAantigen polypeptide), it is typically located at a site 5′ from thepromoter. The enhancer may be native to the non-HLA antigenpolynucleotide sequence or may be heterologous to the non-HLA antigenpolynucleotide sequence.

The transformation of an expression vector encoding a non-HLA antigenpolypeptide (or HLA antigen polypeptide) into a host cell may beaccomplished by well-known methods such as transfection, infection,calcium chloride, electroporation, microinjection, lipofection or theDEAE-dextran method or any other technique known in the art. Thesemethods and other suitable methods are well known in the art, forexample, in Sambrook, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press; 3rd ed., 2001, the disclosure of whichis incorporated herein by reference in its entirety.

Expression vectors of the invention may be constructed from a startingvector such as a commercially available vector. Such vectors may or maynot contain all of the desired flanking sequences. Where one or more ofthe desired flanking sequences are not already present in the vector,they may be individually obtained and ligated into the vector. Methodsused for obtaining each of the flanking sequences are well known to oneskilled in the art.

Preferred vectors for practicing this invention are those which arecompatible with bacterial, insect, and mammalian host cells. Suchvectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (InvitrogenCompany, Carlsbad, Calif.), pBSII (Stratagene Company, La Jolla,Calif.), pET15? (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech,Piscataway, N.J.), pEGFP-N2 (Clontech, Palo Alto, Calif.), pETL(BlueBacIl; Invitrogen), pDSR-alpha (PCT Publication No. WO90/14363) andpFastBacDual (Gibco/BRL, Grand Island, N.Y.).

Additional suitable vectors include, but are not limited to, cosmids,plasmids or modified viruses, but it will be appreciated that the vectorsystem must be compatible with the selected host cell. Such vectorsinclude, but are not limited to plasmids such as Bluescript® plasmidderivatives (a high copy number ColE1-based phagemid, Stratagene CloningSystems Inc., La Jolla Calif.), PCR cloning plasmids designed forcloning Taq-amplified PCR products (e.g., TOPO™ TA Cloning® Kit, PCR2.1®plasmid derivatives, Invitrogen, Carlsbad, Calif.), and mammalian,yeast, or virus vectors such as a baculovirus expression system (pBacPAKplasmid derivatives, Clontech, Palo Alto, Calif.). The recombinantmolecules can be introduced into host cells via transformation,transfection, infection, or other known techniques.

Host cells may be prokaryotic host cells (such as E. coli) or eukaryotichost cells (such as a yeast cell, an insect cell or a vertebrate cell).The host cell, when cultured under appropriate conditions, synthesizes anon-HLA antigen polypeptide (or HLA antigen polypeptide) describedherein which can subsequently be collected from the culture medium (ifthe host cell secretes it into the medium) or directly from the hostcell producing it (if it is not secreted). The selection of anappropriate host cell will depend upon various factors, such as desiredexpression levels, polypeptide modifications that are desirable ornecessary for activity, such as glycosylation or phosphorylation, andease of folding into a biologically active molecule.

A number of suitable host cells are known in the art and many areavailable from the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209. Examples include, butare not limited to, mammalian cells, such as Chinese hamster ovary cells(CHO) (ATCC No. CCL61) CHO DHFR-cells (Urlaub et al., Proc. Natl. Acad.Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293 or 293Tcells (ATCC No. CRL1573), Hmy2.ClR cells (ATCC No. CRL1992) or K562cells (ATCC No. CCL243). The selection of suitable mammalian host cellsand methods for transformation, culture, amplification, screening andproduct production and purification are known in the art. Other suitablemammalian cell lines, are the monkey COS-1 (ATCC No. CRL1650) and COS-7cell lines (ATCC No. CRL1651), and the CV-1 cell line (ATCC No. CCL70).Further exemplary mammalian host cells include primate cell lines androdent cell lines, including transformed cell lines. Normal diploidcells, cell strains derived from in vitro culture of primary tissue, aswell as primary explants, are also suitable. Candidate cells may begenotypically deficient in the selection gene, or may contain adominantly acting selection gene. Other suitable mammalian cell linesinclude but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHKor HaK hamster cell lines, which are available from the ATCC. Each ofthese cell lines is known by and available to those skilled in the artof protein expression.

Methods of Detecting HLA- and Non-HLA-Specific Antibodies

The invention provides for methods for determining the percentage ofpanel reactive antibodies in a biological sample from a subject againsthuman leukocyte antigens. In some embodiments, the method comprisescontacting a first collection of solid-phase substrates subtypes and asecond collection of solid-phase substrate subtypes with serum from saidsubject for a sufficient time for anti-HLA antibodies in said serum tobind to said HLA-antigens to form a complex, wherein each substratesubtype in the first collection is coated with different purified HLAantigens to present HLA antigens derived from a cell population of asingle cell, wherein each substrate subtype of the second collection iscoated with different purified non-HLA antigens listed in Table 1 orTable 1A, detecting the presence of the complex to determine thepresence or absence of panel reactive antibodies, and determining thepercentage of panel reactive antibodies in the serum.

The term “panel reactive antibody” as used herein refers to an antibodyin the biological sample from a subject that specifically binds to anHLA antigen present on the solid-phase substrate or specifically bindsto a non-HLA antigen.

The methods are carried out with solid-phase panels wherein the panelcomprises substrates that present (or have immobilized) at least one ormore selected HLA antigens. The invention also may be carried out withliquid-phase assays such as assays using column chromatography, affinitychromatography, thin layer chromatography, liquid-phase immunodiagnostic(LIPA) assays, liquid-phase chemiluminescent ELISA and liquid-phaseimmunoradiometric (IRMA) to name a few.

HLA- and non-HLA antigens described herein may be a whole protein, atruncated protein, a fragment of a protein or a peptide. Antigens may benaturally occurring, genetically engineered variants of the protein, ormay be codon optimized for expression in a particular mammalian subjector host. Generally, a B-cell epitope will include at least about 5 aminoacids but can be as small as 3-4 amino acids. The antigens may berecombinantly expressed and purified from cells that either endogenouslyexpress the HLA antigens at a low level or do those that do notendogenously express the HLA antigens. Furthermore, the HLA antigens maybe recombinantly expressed and presented on the cell surface, and thecells would be used in the methods of the invention.

Normally, an epitope will include between about 7 and 15 amino acids,such as, 9, 10, 12 or 15 amino acids. The term “antigen” denotes bothsubunit antigens, (i.e., antigens which are separate and discrete from awhole organism with which the antigen is associated in nature).Antibodies such as anti-idiotype antibodies, or fragments thereof, andsynthetic peptide mimotopes, that is synthetic peptides which can mimican antigen or antigenic determinant, are also captured under thedefinition of antigen as used herein.

Furthermore, for purposes of the present invention, an “antigen” refersto a protein, which includes modifications, such as deletions, additionsand substitutions, generally conservative in nature, to the naturallyoccurring sequence, so long as the protein maintains the ability toelicit an immunological response, as defined herein. These modificationsmay be deliberate, as through site-directed mutagenesis, or may beaccidental, such as through mutations of hosts which produce theantigens. Antigens of the present invention may also be codon optimizedby methods known in the art to improve their expression orimmunogenicity in the host.

Exemplary solid-phase assays such as assays of the invention may usesolid substrates such as microparticles, microbeads, magnetic particlessuch as ferromagnetic beads and paramagnetic beads, microtiter plates,membranes, filters, glass, metal, metal-alloy, anopol, polymers, nylon,plastic or microarrays such as protein chips. Microarrays may be of anymaterial such as glass or silica. Binding on a microtiter plate may bedetected using ELISA assays, RIA assays or other immunosorbent sandwichassays. Binding on a filter may be detected using immunoblottingtechniques.

Methods known in the art for HLA testing include thecomplement-dependent lymphocytotoxicity (CDC) test in which serum from arecipient is incubated with donor or panel lymphocytes followed byincubation with complement. The level of cytotoxicity is estimated bydiscriminating between dead and viable cells using a dye. This method islabor intensive, requires viable cells, may be nonspecific and requiresa subjective evaluation.

Pouletty et al. U.S. Pat. No. 5,223,397 discloses methods for testingHLA compatibility between a donor and a recipient comprising the stepsof adding blood from the donor to a substrate having anti-HLA antibodiesbound thereto and incubating for sufficient time for soluble HLAantigens present in the blood to bind to the antibodies or ligand. Bloodfrom the recipient is then added to the solid substrate whereby anyantibody specific for any HLA antigens bound to the solid substrate maybecome bound. The detection of an absence of antibodies from therecipient's blood to the HLA antigen is indicative of a cross-match.

Zaer et al., Transplantion 63: 48-51 (1997) discloses use of an ELISAusing HLA class I molecules purified from pooled platelets to detectanti-HLA antibodies. The reference reports that in patients found to beunsensitized, the incidence of false-positive results was less for ELISAtesting than for panel studies. In patients who were highly sensitized,both tests performed equally well, whereas discordant results wereregistered mainly in cases of mild sensitization. In such cases, theincidence of false-negative results was higher for ELISA testing thanfor panel studies.

Of interest to the present invention are assay methods making use offlow cytometry. Wilson et al., J. Immunol. Methods 107: 231-237 (1988)disclose the use of polyacrylamide microspheres coupled with cellmembrane proteins in immunofluorescence assays for antibodies tomembrane-associated antigens. The method is said to make possible therapid flow cytometric analysis of plasma membrane antigens from cellpopulations that would otherwise be unsuitable for use in flowcytometry. Scillian et al., Blood 73: 2041-2048 (1989) disclose the useof immunoreactive beads in flow cytometric assays for detection ofantibodies to HIV. Frengen et al., Clin. Chem. 40/3: 420-425 (1994)disclose the use of flow cytometry for particle-based immunoassays ofce-fetoprotein (AFP). This reference further reports the ability ofserum factors to cross-link labeled mouse monoclonal antibodies ofirrelevant specificity to different particle types coated with variousimmunoglobulins.

Flow cytometry methods using lymphocytes are also known but suffer withdifficulties because of the activity of auto-antibodies. See Shroyer etal., Transplantation 59:626-630 Moreover, when using flow cytometry withlymphocytes, use of ten or more different lymphocytes tends to result inconfusing signals. As a consequence, studies using lymphocytes have beenlimited by presenting a small panel of HLA antigens that do noteffectively simulate the distribution of HLA antigens in a normal humanpopulation.

Sumitran-Karuppan et al., Transplantation 61: 1539-1545 (1996) disclosesthe use of magnetic beads which use an anti-HLA capture antibody toimmobilize a variety of soluble HLA antigens pooled from 80 to 100individuals on each bead. The beads can then be directly added topatient serum for efficient absorption of HLA antibodies. The referencediscloses visualization of antibody binding to the antigen-coated beadsusing flow cytometry. The reference suggests that this development willallow testing for antibody specificity for crossmatching purposes andfor the screening of panel-reactive antibodies. The methods ofSumitran-Karuppan are limited, however, because the pooling of antigenscauses sensitivity to certain rare HLA antigens. Moreover, the method isnot capable of detecting the percentage of PRA.

Solid-Phase Substrates

The solid-phase substrates described herein include, but are not limitedto, microparticles, microbeads, magnetic beads, ion torrent beads, flowcytometry beads, beads or microspheres of any material, e.g. silica,gold, latex, polymers such as polystyrene, polysulfone and polyethyl, orhydrogel. The solid-phase substrate may also be an affinity purificationcolumn. Additional exemplary microparticles are encoded with the dyesand the antigens are immobilized to the encoded microparticles. Themicroparticles used in the methods of the invention are commerciallyavailable from sources such from Luminex Inc., Invitrogen (Carlsbad,Calif.), Polysciences Inc. (Warrington, Pa.) and Bangs Laboratories(Fishers, Ind.) to name a few.

In some embodiments, the solid-phase substrate is a microbead. Themicrobead, in some embodiments, has a diameter ranging from about 2 μmto about 15 μm, inclusive of each endpoint of the range. Microbeadshaving a diameter of about 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9μm, 10 μm, 11 μm, 12 μm, 13 μm, 14 μm or 15 μm are also contemplated.

The solid-phase substrates described herein may comprise a detectablelabel or another identifying characteristic. The solid-phase substratesmay comprise a single fluorescent dye or multiple fluorescent dyes. Inone embodiment, the microparticles are internally labeled withfluorescent dyes and contain surface carboxyl groups for covalentattachment of biomolecules. In another embodiment, the solid-phasesubstrates are internally labeled with fluorescent dyes and contain asurface layer of Avidin for near covalent binding of biotin andbiotinylated ligands. In another embodiment, the solid-phase substratesmay comprise a combination of different dyes, such as a fluorescent anda non-fluorescent dye. For example, the microparticles may be labeledwith E)-5-[2-(methoxycarbonyl)ethenyl]cytidine, which is anonfluorescent molecule, that when subjected to ultraviolet (UV)irradiation yields a single product,3-?-D-ribofuranosyl-2,7-dioxopyrido[2,3-d]pyrimidine, which displays astrong fluorescence signal. In another embodiment, the solid-phasesubstrates may comprise bar codes as an identifiable characteristic asdescribed in U.S. Patent Publication No. US 20070037195.

In another embodiment, the solid-phase substrate may be nanocrystals orquantum dots. These nanocrystals are substances that absorb photons oflight, then re-emit photons at a different wavelength (fluorophores). Inaddition, additional florescent labels, or secondary antibodies may beconjugated to the nanocrystals. These nanocrystals are commerciallyavailable form sources such as Invitrogen and Evident Technologies(Troy, N.Y.),

The invention can be carried out with any system that detects theidentifiable characteristic or label, such as FLOW cytometry. Detectionof fluorescent labels may also be carried out using a microscope orcamera that will read the image on the microparticles, such as theBioarray BeadChip (Bioarray Solutions, Ltd., Warren, N.J.). The BeadChipformat combines microparticle (“bead”) chemistry with semiconductorwafer processing in which binding to the microparticle is recorded usingan optical microscope and camera.

Biological samples for use in the methods described herein include, butare not limited to, whole blood, blood derivatives, red blood cellconcentrates, plasma, serum, fresh frozen plasma, whole blood derivedplatelet concentrates, apheresis platelets, pooled platelets,intravenous gamma-globulin, cryoprecipitate, cerebrospinal fluid,tissues and cells such as epithelial cells, such as those collected fromthe buccal cavity, stem cells, leukocytes, neutrophils and granulocytes.The biological samples may be obtained from a human donor of tissue orcells intended for transplantation or a human donor of blood or bloodderivatives intended for transfusion. The biological sample may beobtained from a healthy bone marrow donor or a subject of a paternitytest. The biological sample may also be obtained from a human subjectthat is an intended recipient of a transplant or transfusion, or thehuman subject that is donating the tissue or organ intended fortransplantation or transfusion. Alternatively, the biological sample maybe obtained directly from tissues or cells that are intended fortransplantation in a human recipient. In addition, the biological samplemay be obtained from blood or blood derivatives that are intended fortransfusion in a human recipient. In some embodiments, the sample isobtained before the subject has received the transplant or transfusion.In some embodiments, the sample is obtained after the subject hasreceived the transplant or transfusion. In still further embodiments,the sample is obtained both before and after the subject has receivedthe transplant or transfusion in order to monitor success of thetransplant or transfusion.

Antibodies useful for detecting the antigens described herein may bepolyclonal antibodies, monoclonal antibodies, antibody fragments whichretain their ability to bind their unique epitope (e.g., Fv, Fab andF(ab)₂ fragments), single chain antibodies and human or humanizedantibodies. Antibodies may be generated by techniques standard in theart using an antigenic HLA epitope. See, e.g. Kohler et al., Nature,256:495-497 (1975), Brodeur et al., Monoclonal Antibody ProductionTechniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York,1987). Antibody molecules of the present invention include the classesof IgG (as well as subtypes IgG 1, IgG 2a, and IgG2b), IgM, IgA, IgD,and IgE.

The antibodies of the invention may be labeled for detection of bindingwithin the biological sample. The antibodies may comprise a radioactivelabel such as 3H, 14C, 32P, 35S, or 125I. In addition, the labels may bea fluorescent or chemiluminescent compound, such as fluoresceinisothiocyanate, phycoerythrin, rhodamine, or luciferin. The labels maybe enzymes such as alkaline phosphatase, β-galactosidase, biotin andavidin or horseradish peroxidase (Bayer et al., Meth. Enz., 184:138-163(1990)).

Specific binding of an antibody to an antigen described herein within abiological sample may be carried out using Western blot analysis withimmunoblotting, immunocytochemistry, immunohistochemistry, dot blotanalysis, flow cytometry, ELISA assays or RIA assays. These techniquesand other approaches are conventional in the art (See Sambrook et al.,Molecular Cloning: A Laboratory Manual, cold Springs Harbor Laboratories(New York, 1989).

Kits

The invention also provides for kits to carry out the methods of theinvention. In particular, the invention provides for kit for determiningthe percentage of panel reactive antibodies in serum of a subjectagainst HLA antigens comprising a first collection of solid-phasesubstrates wherein each solid-phase substrate is coated with differentpurified HLA antigens to represent the HLA antigen population of asingle cell line such that said collection simulates the distribution ofHLA antigens in a normal human population and a second collection ofsolid phase substrates wherein each substrate is coated with differentpurified non-HLA antigens listed in Table 1. The antigens provided inthe kit may be conjugated to solid substrates in the kit. Alternatively,the kit comprises solid substrates and antigens and the skilled artisancan conjugate the antigens to the solid substrates allowing foroptimization of the antigens used in the assay. The kits may alsocomprise the reagents necessary to detect and measure antibodies, suchas HLA antibodies for use as a positive control.

In some embodiments, the HLA antigens comprise Class I HLA antigens(e.g., wherein the HLA antigens are selected such that the HLA antigenspresented on the solid phase substrate comprise Class I HLA antigens soas to simulate the distribution of Class I HLA antigens in a normalhuman population). In some embodiments, the HLA antigens comprise ClassII HLA antigens.

In some embodiments, the first collection comprises 54 different Class IHLA antigens, optionally purified from 30 different cell lines. In otherembodiments, the first collection comprises 22 different Class II HLAantigens.

In some embodiments, the non-HLA antigens in the second collection areoptionally a fusion protein comprising at least one domain, wherein thedomain is a signal peptide, a modified cytoplasmic domain, purificationtag or detection tag. In some embodiments, domain is the B2 signalpeptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag.

The kits described herein may further comprise any components necessaryto carry out the detection assays that are conventional in the art. Forexample, the kits may comprise buffers, loading dyes, gels such aspolyacrylamide gels and molecular weight markers for preparing SDS-PAGEgels to carry out Western blots. The kits may also comprise filters,membranes blocking buffers, control buffers, isotype control antibodies,wash buffers or buffers and reagents for detection to carry outimmunoblotting or dot blotting analysis such as labeled secondaryantibodies. The kit may also comprise fixing reagents, blocking buffers,control buffers, wash buffers, staining dyes and detection reagentsincluding anti-idiospecific antibodies. Furthermore, the kits maycomprise the necessary reagents and tools to carryout flow cytometry,ELISA assays, RIA assays or microtoxicity assays.

Other aspects and advantages of the present invention will be understoodupon consideration of the following illustrative examples.

EXAMPLES Example 1 A Multiplex Assay on a Panel Consisting of 10 Non-HLAAntigens in One Single Test

Panel consists of 10 non-HLA were incubated with 4 different patientserum. Sera are used neat. The microbeads are subsequently washed withwash buffer comprising PBS with 0.1% polysorbate 20 (TWEEN) andincubated with goat anti-human IgG antibodies conjugated withphycocrythrin (PE) for 30 minutes. The microbeads were washed two timeswith wash buffer and analyzed on a Luminex analyzer according to themanufacturer's instructions.

Reaction pattern is compared. Four individuals (S 10823K, S11114A,S11143B and FL71681) showed distinct reaction patterns on 10 non-HLAantigens tested (FIG. 1).

Example 2 Trend Increase in Anti-non-HLA Allosera in a Lung TransplantRecipient During 1^(st) Graft Rejection on a Panel Consisting of 58Non-HLA Antigens in One Single Test

Panel consists of 58 non-HLA were incubated with 4 serum samplescollected from different stage of graft rejection. Sera are used neat.The microbeads are subsequently washed with wash buffer comprising PBSwith 0.1% polysorbate 20 (TWEEN) and incubated with goat anti-human IgGantibodies conjugated with phycocrythrin (PE) for 30 minutes. Themicrobeads were washed two times with wash buffer and analyzed on aLuminex analyzer according to the manufacturer's instructions.

Reaction pattern is compared on each individual non-HLA and analyzedagainst in a time course plot. Trend line is determined. Six non-HLAantigens show a correlation over the increasing anti-allosera activitieswith the graft rejection progression (FIG. 2).

Example 3 Increase of Anti-non-HLA Allosera in Transplant RecipientsDuring Graft Rejection on a Panel Consisting of 21 Non-HLA Antigens inOne Single Test

Panel consists of 21 non-HLA were incubated with serum samples collectedfrom 13 graft recipients for graft post-transplant monitoring. Sera arecollected on time course. Sera are used neat. The microbeads aresubsequently washed with wash buffer comprising PBS with 0.1%polysorbate 20 (TWEEN) and incubated with goat anti-human IgG antibodiesconjugated with phycocrythrin (PE) for 30 minutes. The microbeads werewashed two times with wash buffer and analyzed on a Luminex analyzeraccording to the manufacturer's instructions.

Reaction pattern is compared on each individual non-HLA antigens andeach individual graft recipient is analyzed against on a time courseplot. Trend line on each non-HLA antigen over individual patient isdetermined. Six non-HLA antigens show a correlation over the increasinganti-allosera activities with the graft rejection progression amonggraft recipient monitored. See Table 2.

TABLE 2 % Patient shows positive increase of non-HLA allosera duringpost-translation non-HLA Antigen monitoring TubA1B 0.23 Perlican 0.69PRKRIP1 0.31 EDNRA 0.23 FLRT2 0.38 Vimentin 0.15

Example 4 Increase Detection Sensitization by Fusion Tag in One SingleTest

Enhancing antigenic polypeptide NusA-V5 binding on Luminex beads by asynthetic domain, DPD. The antigenic polypeptides NusA-V5 were fused toDPD. Mouse anti V5 antibody (1 μg) are incubated with NusA polypeptidescontaining microbeads. The NusA polypeptides containing microbeads aresubsequently washed with wash buffer comprising PBS with 0.1%polysorbate 20 (TWEEN) and incubated with goat anti-mouse IgG antibodiesconjugated with phycocrythrin (PE) for 30 minutes. The microbeads werewashed two times with wash buffer and analyzed on a Luminex analyzeraccording to the manufacturer's instruction.

The NusA-V5 fused with DPD tag shows higher sensitivity compared with nofusion (FIG. 3).

Example 5 Box and Whiskers' Plot of Non-HLA Antigens from Two PatientPopulation Using a Panel of 21 Non-HLA Antigens in One Test

Panel consists of 21 non-HLA were incubated with serum samples collectedfrom graft recipients before and after transplant for graftpost-transplant monitoring. Sera are collect and used neat. Themicrobeads are subsequently washed with wash buffer comprising PBS with0.1% polysorbate 20 (TWEEN) and incubated with goat anti-human IgGantibodies conjugated with phycocrythrin (PE) for 30 minutes. Themicrobeads were washed two times with wash buffer and analyzed on aLuminex analyzer according to the manufacturer's instructions.

Serum anti non-HLA alloantibodies activities are determined for 45post-transplant allograft patients and 33 pre-transplant allograftpatients. The median (line) and IQR (box top and bottom) values areshown with a Mann-Whitney Rank Sum test providing a p-value listed inthe box. Three non-HLA antigens show a significance increase of de novoalloantibodies (FIG. 4).

Example 6 Antibodies to Non-HLA Antigens have been Identified in KidneyAllograft Patients

Anti-vimentin IgG and IgM which target at the non-HLA antigen vimentinhas been identified in a chronic kidney transplant patient underpost-transplant monitoring. In addition, high titer of HLA IgMantibodies was observed (Table 3)

TABLE 3 Large scale monitoring of both nHLA and non-HLA antigens SignalsMonitoring Vimentin HLA time IgG IgM IgG IgM beginning 708 2910 175 340  1 month 699 3579 172 204   1 year 1281 4537 141 367 1.5 year 1080 7256139 1025

Example 7

According to this example, Class I HLA antigen preparations werepurified from Epstein Barr virus transformed lymphocyte cell linesaccording to the methods of Henderson et al., Virology 76: 152-163(1977). Thirty of the Class I HLA antigen preparations were thenselected to simulate the distribution of HLA in a normal population asset out in Table 4 and were coated by passive absorption onto 3 μm latexbeads obtained from Spherotech according to the method of Cantarero etal., Anal. Biochem., 105: 373-382 (1980).

TABLE 4 Bead No. HLA CLASS I Antigen Typing 1 A11 B27, 48 2 A2, 29 B39,56 3 A1, 29 B8, 45 4 A2, 24 B7, 55 5 A2, 25 B18, 64 6 A26, 24 B52, 62 7A31, 68 B53 8 A2, 11 B13, 62 9 A23, 33 B45, 63 10 A23, 34 B44 11 A11, 23B49, 52 12 A11, 24 B59, 60 13 A24, 33 B44, 51 14 A23, 26 B41, 72 15 A3,32 B50, 56 16 A2, 24 B54, 67 17 A2 B52, 73 18 A26, 66 B38, 75 19 A11, 33B51, 54 20 A30 B13, 72 21 A30, 36 B35, 71 22 A69 B35, 61 23 A1, 32 B60,64 24 A2 B7, 46 25 A30 B42 26 A2 B8, 58 27 A2, 3 B58, 65 28 A1, 36 B37,57 29 A3, 68 B7, 65 30 A33, 36 B53, 61

The reactivity of the HLA antigen on each bead was confirmed by a panelof serologically defined HLA monoclonal antibodies or by human alloserausing a flow cytometry test. Each bead reacted specifically to the HLAmonoclonal antibodies or allosera with the same HLA specificity.

The sensitivity of the beads was tested by mixing two beads withdifferent typing at different percentages. A minimum of 2 to 3% of onekind of bead was found to be sufficient to detect the antigen.

Example 8

According to this example, the sensitivity of the microbeads useful withthe invention was tested by carrying out a serial dilution of selectedPRA sera. The results presented in Table 5 below show that most PRA seradecrease the percentage of reactivity at a 1:10 dilution measured by acytotoxicity test while they did not decrease the percentage ofreactivity at a 1:40 dilution by use of the microbeads in a flowcytometry device according to the invention.

TABLE 5 Percentage Flow Sera ID Dilution Cytotoxicity Cytometry N21 1 40— 1:10 10 41 1:20 0 30 1:40 0 41 1:50 0 18 1:160 0 16 A2 1 30 1:20 0 251:40 0 26 1:80 0 8 S193 1 25 1:10 31 28 1:20 17 100 1:40 10 100 1:80 0100 S176 1 54 1:10 24 40 1:20 28 41 1:40 10 40 1:50 0 40 S199 1 100 1:1010 97 1:20 3 97 1:40 10 97 1:50 3 99 B73 1 65 1:10 27 54 1:20 3 40 1:403 43 1:50 0 25

Example 9

According to this example, an assay to detect panel reactive antibodieswas carried out by mixing 10 μl of a mixture of the 30 different typesof beads produced according to Example 7 with 100 μl (1:10 diluted)serum to be tested and incubating for 30 minutes at 20-25° C. withgentle rotating. The beads were then washed three times with 1 mL ofwash buffer. The beads were then incubated with 100 μl of 1:100 dilutedGoat anti-human IgG-PE obtained from Jackson InnumoResearch for 30minutes. The beads were then washed twice and 1 mL of wash buffer andread on a flow cytometer (B.D. FacStar Plus). The percentage of PRA isrepresented by the percentage of microbeads which are positivelylabeled.

According to this example, 61 sera samples including 22 negative and 39PRA patients who had panel reactive antibody activities developed byearlier transplantation or transfusion were tested with the resultsshown in FIG. 6 which shows the correlation of the flow cytometryresults with those where the same samples were tested bycomplement-dependent lymphocytotoxicity. The correlation coefficient Ris 0.94 for the 61 data points indicating a high degree of correlationbetween results obtained by flow cytometry and those obtained by acytotoxicity test.

Example 10

According to this example, 30 Class II HLA antigen preparations as setout in Table 5 were purified from Epstein Barr virus transformedlymphocyte cell lines according to the methods of Henderson et al.,Virology 76: 152-163 (1977). The antigen preparations may then be coatedby passive absorption onto 5 μm latex beads obtained from Spherotechaccording to the method of Cantarero et al., Anal. Biochem., 105:373-382 (1980). From this collection of Class II HLA preparations, from15 to 30 beads may selected to simulate the distribution of the 22 ClassII HLA antigens in a normal population.

TABLE 5 Bead No. HLA CLASS II Antigen Typing Typing 1 DR15, 9 53, 51DQ5, 9 2 DR4, 15 53, 51 DQ6, 7 3 DR16, 4 53, 51 DQ4, 5 4 DR8, 14 52 DQ4,5 5 DR4, 7 53 DQ2, 8 6 DR15, 18 51, 52 DQ6, 4 7 DR11, 12 52 DQ5, 7 8DR103, 17 52 DQ5, 2 9 DR1, 13 52 DQ5, 6 10 DR9, 10 53 DQ5, 9 11 DR15, 1251, 52 DQ5, 7 12 DR16, 14 51, 52 DQ5 13 DR13, 8 52 DQ5, 6 14 DR11, 13 52DQ5, 6 15 DR17, 7 52, 53 DQ2, 9 16 DR15, 8 51 DQ6, 8 17 DR15, 4 51, 53DQ2, 6 18 DR15, 17 51, 52 DQ6, 2 19 DR15, 7 51, 53 DQ6, 2 20 DR1, 7 53DQ2, 5 21 DR15, 11 52 DQ5, 6 22 DR7, 13 52, 53 DQ6, 9 23 DR15, 13 51, 52DQ6, 2 24 DR9, 14 52, 53 DQ5, 9 25 DR8, 9 53 DQ2, 7 26 DR17, 14 52 DQ2,5 27 DR1, 11 52 DQ5, 6 28 DR17, 4 52, 53 DQ2 29 DR11, 4 52, 53 DQ7, 8 30DR1, 14 52 DQ5

Example 11

According to this example, 3 μm latex beads presenting HLA Class Iantigens produced according to the methods of Example 7 and 5 μ.m latexbeads presenting HLA Class II antigens produced according to the methodsof Example 9 were mixed to perform an assay to detect the presence ofantibodies specific to HLA Class I and Class II antigens. Because thebeads presenting HLA Class II antigens are different in size from theHLA Class I beads, the two different sized beads can be electronicallydistinguished according to their sizes when analyzed on a flow cytometeras illustrated in FIGS. 7A-7D. FIGS. 7A-7D depict the reaction of themixture of Class I and Class II beads and their reaction to anti-HLAClass I antibodies (FIGS. 7A and 7B) or anti-HLA Class II antibodies(FIGS. 7C and 7D). When the Class I beads are selected by gating aroundthe 3 μm size, the beads react to the anti-Class I antibody asillustrated in FIG. 7A. When the Class II beads are selected by gatingaround the 5 μm size, there is no reaction to the anti-Class I antibodyas illustrated in FIG. 7B. The reaction pattern of the mixed beads tothe anti-class II antibody is the reverse. When Class I beads areselected by gating around 3 μm in size, the beads do not react to theanti-Class II antibody as illustrated in FIG. 7C. When Class IIantibodies are selected by gating around 5 μm in size, the Class IIantigen beads react to the anti-Class II antibody as illustrated in FIG.7D.

Numerous modifications and variations in the practice of the inventionare expected to occur to those skilled in the art upon consideration ofthe foregoing description on the presently preferred embodimentsthereof. Consequently, the only limitations which should be placed uponthe scope of the present invention are those that appear in the appendedclaims.

1. A composition comprising a first collection of solid-phase substrateseach coated with a different purified human leukocytes antigen (HLA) torepresent the HLA antigen population of a single cell line and a secondcollection of solid-phase substrates each coated with at a differentnon-HLA antigen listed in Table 1 or Table 1A.
 2. The composition ofclaim 1 wherein the different purified HLA antigens are Class I HLAantigens or Class II HLA antigens.
 3. (canceled)
 4. The composition ofclaim 1, wherein the non-HLA antigen is a fusion protein comprising atleast one domain, wherein the domain is a signal peptide, a modifiedcytoplasmic domain, purification tag or detection tag.
 5. Thecomposition of claim 4 wherein the domain is the B2 signal peptide, HLAcytoplasmic domain, EK Tag, V5 Tag or DPD Tag. 6-10. (canceled)
 11. Thecomposition of claim 1, wherein each solid phase substrates isdetectably distinguishable the other solid-phase substrates within thecomposition.
 12. (canceled)
 13. A kit for determining the percentage ofpanel reactive antibodies in serum of a subject against HLA antigenscomprising a first collection of solid-phase substrates wherein eachsolid-phase substrate is coated with different purified HLA antigens torepresent the HLA antigen population of a single cell line such thatsaid collection simulates the distribution of HLA antigens in a normalhuman population and a second collection of solid phase substrateswherein each substrate is coated with different purified non-HLAantigens listed in Table 1 or Table 1A.
 14. (canceled)
 15. The kit ofclaim 13 wherein the HLA antigens are selected such that the HLAantigens presented on the solid phase substrate comprise Class I HLAantigens so as to simulate the distribution of Class I HLA antigens in anormal human population.
 16. (canceled)
 17. The kit of claim 13 whereinthe first collection comprises 54 different Class I HLA antigens. 18.The kit of claim 17 wherein the 54 different Class I HLA antigens arepurified from 30 different cell lines.
 19. The kit of claim 13 whereinthe first collection comprises 22 different Class II HLA antigens. 20.The kit of claim 13 wherein the non-HLA antigen is a fusion proteincomprising at least one domain, wherein the domain is a signal peptide,a modified cytoplasmic domain, purification tag or detection tag. 21.The kit of claim 20 wherein the domain is the B2 signal peptide, HLAcytoplasmic domain, EK Tag, V5 Tag or DPD Tag.
 22. (canceled) 23.(canceled)
 24. The kit of claim 13, wherein at least one solid-phasesubstrate is detectably distinguishable from at least one other solidphase substrate. 25-29. (canceled)
 30. The kit of claim 22 wherein saidfirst collection comprises at least one 3 μm microbead presenting ClassI HLA antigens and at least one 5 μm microbead presenting Class II HLAantigens.
 31. A method for determining the percentage of panel reactiveantibodies in serum of a subject against human leukocyte antigens (HLA)antigens, said method comprising: a. contacting a first collection ofsolid-phase substrates subtypes and a second collection of solid-phasesubstrate subtypes with serum from said subject for a sufficient timefor anti-HLA antibodies in said serum to bind to said HLA-antigens toform a complex, wherein each substrate subtype in the first collectionis coated with different purified HLA antigens to present HLA antigensderived from a cell population of a single cell, wherein each substratesubtype of the second collection is coated with different purifiednon-HLA antigens listed in Table 1 or Table 1A, b. detecting thepresence of the complex to determine the presence or absence of panelreactive antibodies, and c. determining the percentage of panel reactiveantibodies in the serum.
 32. The method of claim 31 wherein thedetecting step comprises detecting labeled ligand bound to the complexto determine the presence or absence of panel reactive antibodies. 33.(canceled)
 34. The method of claim 31 wherein the detection stepcomprises detecting the presence of the complex using a solid phaseimmunoassay or a multiplexed bead immunoassay. 35-38. (canceled)
 39. Themethod of claim 31, wherein the first collection of substrates isselected such that the HLA antigens presented thereon simulatedistribution of Class I HLA antigens in a normal human population. 40.The method of claim 31, wherein said first collection of substratescomprises 54 different Class I HLA antigens.
 41. The method of claim 31,wherein said first collection of substrates comprises 54 different ClassI HLA antigens purified from 30 different cells. 42-43. (canceled) 44.The method of claim 31, wherein the non-HLA antigen is a fusion proteincomprising at least one domain, wherein the domain is a signal peptide,a modified cytoplasmic domain, purification tag or detection tag. 45.The composition of claim 44, wherein the domain is the B2 signalpeptide, HLA cytoplasmic domain, EK Tag, V5 Tag or DPD Tag.
 46. Themethod of claim 31, wherein each solid-phase substrate is detectablydistinguishable from the other solid phase substrates within acollection. 47-49. (canceled)
 50. The method of claim 35, wherein saidmicrobeads comprise a mixture of 3 μm microbeads presenting Class I HLAantigens and 5 μm microbeads presenting Class II HLA antigens.
 51. Themethod of claim 31, wherein the subject is a transplant or transfusionrecipient.
 52. The method of claim 31, wherein the serum sample iscollected before the subject has received a transplant or transfusion.53. The method of claim 31, wherein the serum sample is collected afterthe subject has received a transplant or transfusion.